Immunoassays for lamotrigine

ABSTRACT

Generally, the present invention relates to lamotrigine analogs that have substituents at the triazine 3-position and on the benzene 4-position and 5-position. The lamotrigine analogs can include immunogenic moieties that can be used to prepare anti-lamotrigine antibodies, or antigenic moieties that can be used in immunodiagnostic assays for lamotrigine. Also, the lamotrigine analog can include tracer moieties for detecting the presence or amount of the analog during an immunodiagnostic assay. Additionally, the lamotrigine analogs can be used in immunodiagnostic assays to compete with lamotrigine for binding with anti-lamotrigine antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This U.S. patent application is a divisional of U.S. patent applicationhaving Ser. No. 11/254,637 filed on Oct. 20, 2005 now U.S. Pat. No.7,678,551 with Anlong Ouyang, Ph.D. et al. as inventors and entitled,“IMMUNOASSAYS FOR LAMOTRIGINE,” which claims the benefit of U.S.Provisional Application having Ser. No. 60/621,764, entitled,“IMMUNOASSAYS FOR LAMOTRIGINE,” which was filed on Oct. 25, 2004, withAnlong Ouyang, Ph.D. et al. as inventors, the entireties of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. The Field of the Invention

The present invention relates to lamotrigine immunodiagnostic reagentsand protocols. More particularly, the present invention relates tolamotrigine, lamotrigine analogs, immunogens and antigens prepared fromlamotrigine analogs, antibodies prepared from lamotrigine-basedimmunogens, and methods of making and using the same.

2. The Related Technology

Lamotrigine, chemically represented as3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine and shown below, is ananti-epileptic drug (“AED”) of the phenyltriazine class, and ischemically unrelated to existing AEDs. Lamotrigine is the activeingredient in LAMICTAL® (Glaxo Wellcome), an FDA-approved drug used foranti-epileptic treatment as well as for treatment of the psychiatricdisorders, such as bipolar disease.

Epilepsy is brain function disorder that results in repeated seizures.Lamotrigine has been shown to have a broad spectrum of clinicalefficacy, and is effective in treating and/or preventing partialseizures, primary and secondarily generalized seizures, absenceseizures, and drop attacks associated with Lennox-Gastaut syndrome.

It is well known that various drugs such as AEDs, can have differentpharmacokinetic and/or pharmacodynamic profiles in different patientpopulations, which results in the therapeutic drug monitoring (“TDM”) ofAEDs to be vitally important. One goal of a TDM program is to optimize apatient's clinical outcome by managing and/or optimizing a medicationregimen with the assistance of determining drug concentrations atvarious times. Accordingly, the drug dose and regimen can be modulatedfor a single patient or patient population based on TDM.

Several characteristics of lamotrigine suggest there is a clinical needto individualize patient therapy by use of TDM. It has been suggestedthat there are large inter-individual variations in dose versus serumconcentrations in patients, and pharmacokinetic variability plays amajor role in the lamotrigine dosage requirements needed to achieveoptimum serum concentrations.

It as been suggested that an appropriate range of optimal serumconcentrations for lamotrigine would be 12 to 55 μmol/L in patients withrefractory epilepsy. See Morris R G et al, Br J Clin Pharmacol;46:547-51 (1998). In the responders (>50% seizure reduction), the medianlamotrigine concentration was 31 μmol/L (range, 8-60 μmol/L) comparedwith 62 μmol/L (range, 31-60 μmol/L) in patients with side effects. Assuch, a target range of 10 to 60 μmol/L (2.54-15.24 μg/mL) is nowsuggested for lamotrigine. Thus, effective TDM can be used to predictdosing regimens that can obtain appropriate lamotrigine concentrationswithin the therapeutic index.

Many methods have been described for analyzing lamotrigine. Primarily,the methods include HPLC with ultraviolet (“UV”) detection. See, Fraseret al, Ther Drug Monitoring, 17:174-178, 1995; Lensmeyer et al, TherDrug Monitoring, 19:292-300, 1997; Croci et al. Ther Drug Monitoring23:665-668, 2001. In addition, a competitive binding enzyme immunoassay(ELISA) for the measurement of lamotrigine in sera has been reported.See, Sailstad et al, Ther Drug Monitoring, 13:433-442, 1991. However,such methods are impractical for commercial use due to, for example,long sample preparation time, long assay time, high cost, andlabor-intensive procedures. Thus, a simple and fast analytical methodfor measuring lamotrigine plasma levels is needed for effective TDM,which immunoassay techniques are well suited for such analyticalapplications.

Immunoassay techniques have been developed to detect various drugs inbiological samples and are well suited for such commercial analyticalapplications. Accordingly, immunoassays can be used to quickly assessthe amount of a drug and/or drug metabolite in a patient's blood.Examples of immunoassays can include, but not limited to, homogeneousmicroparticle immunoassay (e.g., immunoturbidimetric) or quantitativemicrosphere system (“QMS®”), fluorescence polarization immunoassay(“FPIA”), cloned enzyme donor immunoassay (“CEDIA”), chemiluminescentmicroparticle immunoassay (“CMIA”), and the like.

Accordingly, it would be advantageous to have immunoassays configured todetect lamotrigine in a patient's blood, serum, plasma, and/or otherbiological fluids or samples. Additionally, it would be advantageous tohave lamotrigine analogs for use in such immunoassays, and/orlamotrigine analog-based immunogens for use in producinganti-lamotrigine antibodies.

BRIEF SUMMARY OF THE INVENTION

Generally, the present invention relates to lamotrigine analogs andimmunodiagnostic assays for lamotrigine. The lamotrigine analogs caninclude operative groups, such as: immunogenic moieties that can be usedto prepare anti-lamotrigine antibodies; antigenic moieties that can beused in immunodiagnostic assays for lamotrigine; or tracer moieties thatcan be used in immunodiagnostic assays. Additionally, the lamotrigineanalogs can be used in immunodiagnostic assays to compete withlamotrigine for anti-lamotrigine antibodies.

In one embodiment of the present invention, a lamotrigine analog caninclude a chemical structure of at least one of Formula 1A, Formula 2A,or Formula 3 A.

Additionally, the foregoing chemical structures of Formula 1A, Formula2A, and/or Formula 3A are scaffolds that can include a variety ofmoieties conjugated thereto. As such, the scaffolds can be furtherdefined by the following: (a) L can be one of the group NH, NHCO, or O;(b) W can be a saturated or unsaturated, substituted or unsubstituted,and straight or branched chain of 1-10 carbon or hetero chain atoms; (c)X can be at least one of a bond between W and Y, a substituted orunsubstituted aromatic or aliphatic group having from 1-2 rings, and/ora saturated or unsaturated, substituted or unsubstituted, or straight orbranched chain having 1-10 carbon or hetero chain atoms; (d) Y isselected from the group consisting of aliphatic, alcohol, amine, amide,carboxylic acid, aldehyde, ester, activated ester, aliphatic ester,imidoester, isocyanate, isothiocyanate, anhydride, thiol, alcohol,thiolactone, diazonium, and maleimido groups; and (e) Y—X—W-L- ofFormula 1 is not a 5-succinylamino moiety. Additionally, Y can be alinker group coupled to an operative group.

In one embodiment, a lamotrigine analog in accordance with any of thescaffolds depicted by formulas 1A, 2A, and/or 3A can be characterized bybeing coupled to an immunogenic moiety via appropriate chemistry, toform an immunogen that generates an antibody at a titer sufficient foruse in an immunodiagnostic assay for lamotrigine. Also, it is possiblefor the lamotrigine analog to be coupled to an immunogenic moiety toform an immunogen that generates an antibody that interacts with theantigen and lamotrigine wherein the affinity, specificity, and/oravidity is substantially similar for lamotrigine and the analog and canbe used in competitive binding studies. Additionally, the lamotrigineanalog can be coupled to a tracer moiety and have sufficient solubilityfor use in an immunodiagnostic assay. The analog can also be coupled toan antigen moiety and have sufficient solubility for use in animmunodiagnostic assay. Further, the lamotrigine analog can be stablyloaded onto a particle or microparticle. Furthermore, the lamotrigineanalog can be coupled to an enzyme, enzyme donor, or enzyme acceptor.

One embodiment of the present invention includes an antibody compositionfor use in an immunodiagnostic system for detecting the presence oflamotrigine in a sample. The antibody composition can include ananti-lamotrigine antibody having at least one binding domain, whereinthe antibody is capable of binding lamotrigine and is capable of bindinga lamotrigine analog. Also, the antibody can be present in a titer of atleast about 1:5,000, more preferably at least about 1:10,000, even morepreferably at least about 1:50,000, still more preferably at least about1:100,000, and most preferably at least about 1:300,000. In someinstances it can be preferably to have an antibody titer as low as1:5,000 or as high as 1:300,000.

Additionally, the antibody is a monoclonal antibody and/or a polyclonalantibody. The antibody can have at least one of affinity, specificity,or avidity for a lamotrigine analog compared to lamotrigine that issufficient for use in a homogeneous or heterogeneous immunodiagnosticassay. As such, the interaction between the antibody and the lamotrigineanalog can be at least 50% of at least one of affinity, specificity, oravidity of the antibody for lamotrigine, even more preferably at least70% of at least one of affinity, specificity, or avidity of the antibodyfor lamotrigine, most preferably at least 90% of at least one ofaffinity, specificity, or avidity of the antibody for lamotrigine.Optionally, at least one of affinity, specificity, or avidity of theantibody for a lamotrigine analog is substantially the same as forlamotrigine.

In one embodiment, the present invention includes a system for use in animmunodiagnostic system for detecting the presence of lamotrigine in asample. Such a system can include the lamotrigine analog and theanti-lamotrigine antibody. In one aspect, the lamotrigine analogincludes a linker substituent coupled to an end group selected from thegroup consisting of saturated or unsaturated aliphatics, alcohols,amines, amides, carboxylic acids, aldehydes, esters, activated esters,aliphatic esters, imidoesters, isocyanates, isothiocyanates, anhydrides,thiols, alcohols, thiolactones, diazonium groups, and maleimido groups.In the system, the linker substituent can be characterized by at leastone of the following: (a) a 5-position substituent having at least a 5carbon or hetero atom aliphatic chain; (b) a 4-position substituenthaving at least at least a 4 carbon or hetero atom aliphatic chain; or(c) a 3-position substituent having at least a 4 carbon or hetero atomaliphatic chain. Additionally, one of the lamotrigine analog oranti-lamotrigine antibody can be coupled with one of a particle,magnetic particle, microparticle, microsphere, support, enzyme donor, orenzyme acceptor.

In one embodiment, the system can include at least one of the following:(a) a stock composition of lamotrigine; (b) a series of compositionscontaining lamotrigine at different concentrations, the series ofcompositions forming a concentration gradient; (c) the lamotrigineanalog having a tracer moiety; (d) the lamotrigine analog coupled to amicroparticle; (e) the antibody coupled to a microparticle; (f) thelamotrigine analog having an enzyme donor and a corresponding enzymeacceptor; (g) the lamotrigine analog having to an enzyme acceptor and acorresponding enzyme donor; or (h) the antibody loaded on a particlesuitable for separation by filtration or sedimentation.

The present invention also includes methods of performingimmunodiagnostic assays for detecting the presence of lamotrigine in asample. Such methods can include combining an anti-lamotrigine antibodyat a titer of at least 1:5,000 and a lamotrigine analog with a sampleobtained from a subject previously administered lamotrigine to form afirst composition. Any free lamotrigine from the sample and thelamotrigine analog are then allowed to compete for binding with theantibody. After the competitive binding, the binding between thelamotrigine analog and the antibody is detected.

In one embodiment, the immunodiagnostic assay utilizes a lamotrigineanalog including a fluorescent moiety, and is combined with the antibodyand sample as described. The fluorescent moiety can be excited withpolarized light having a first amount of polarization, and the polarizedlight emitted from the fluorescent moiety having a second amount ofpolarization is detected. Optionally, the first amount of polarizationis compared with the second amount of polarization, and a determinationis made as to whether lamotrigine is present in the sample, wherein thesecond amount of polarization being different from the first amount ofpolarization is an indication that lamotrigine is present in the sample.Additionally, the immunodiagnostic assay can include a control bycombining a known amount of lamotrigine with the lamotrigine analog andantibody to form a control binding composition. The polarized lightemitted from the florescent conjugate in the control binding compositionhaving a third amount of polarization is detected, and compared with thesecond amount of polarization. The amount of lamotrigine present in thesample is then determined.

In one embodiment, an immunodiagnostic assay uses a lamotrigine analogor antibody loaded onto a microparticle. The analog, antibody, andsample are combined into a first composition, where any free lamotriginecompetes with the analog for binding with the antibody. The firstcomposition is then irradiated with incident light, and a firstintensity of light transmitted from the first composition is detected.The minimum intensity of light transmitted from a control bindingcomposition having the lamotrigine analog and antibody and not havingfree lamotrigine is identified and compared with the first intensity ofthe transmitted light. A determination is made as to whether lamotrigineis present in the sample, wherein the minimum intensity being differentfrom the first intensity is an indication that lamotrigine is present inthe sample. Additionally, the immunodiagnostic assay can include acontrol by combining a known amount of lamotrigine with the lamotrigineanalog and antibody to form a control binding composition. The controlbinding composition is then irradiated with incident light, and a secondintensity of light transmitted from the control binding composition isdetected. The amount of lamotrigine present in the sample can then bedetermined, wherein a comparison between the first intensity and thesecond intensity is an indication of the amount of lamotrigine presentin the sample.

In one embodiment, an immunodiagnostic assay uses a lamotrigine analoghaving an enzyme donor. The analog, antibody, and sample are combinedinto a first composition, where any free lamotrigine competes with theanalog for binding with the antibody. An enzyme acceptor and substrateare then combined with the first composition, wherein the substrate iscleavable by interacting with the enzyme donor and enzyme acceptor. Theenzyme activity is then detected. Additionally, the immunodiagnosticassay can include a control by combining a known amount of lamotriginewith the lamotrigine analog and antibody to form a control bindingcomposition, and the enzyme acceptor and substrate are then combinedtherewith. The amount of lamotrigine present in the sample is determinedby a comparison between the enzyme activity and the control enzymeactivity providing an indication of the amount of lamotrigine present inthe sample.

In one embodiment, an immunodiagnostic assay uses a lamotrigine analogthat includes a tracer conjugate. The analog, antibody, and sample arecombined into a first composition, where any free lamotrigine competeswith the analog for binding with the antibody. The antibody is thenseparated from the first composition, and any unbound lamotrigine analogis separated from the antibody. The tracer conjugate bound with theantibody from the competitive binding composition is then detected.Additionally, the immunodiagnostic assay can include a control bycombining a known amount of lamotrigine with the lamotrigine analog andantibody to form a control binding composition. Accordingly, the amountof lamotrigine present in the sample can be determined by a comparisonbetween the amount of tracer conjugate in the first composition and theamount of tracer conjugate in the control binding composition in orderto provide an indication of the amount of lamotrigine present in thesample.

These and other embodiments and features of the present invention willbecome more fully apparent from the following description and appendedclaims, or may be learned by the practice of the invention as set forthhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention willbe rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only typical embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention willbe described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1 is a flow diagram illustrating an embodiment of a method forpreparing an anti-lamotrigine antibody;

FIG. 2 is a flow diagram illustrating an embodiment of a method forperforming an immunodiagnostic assay for lamotrigine;

FIG. 3 is a schematic diagram illustrating an embodiment of acompetitive binding study based on fluorescent polarization;

FIG. 4 is a graph illustrating an embodiment of a calibration curve forlamotrigine;

FIG. 5 is flow diagram illustrating an embodiment of a competitivebinding study based on agglutination;

FIG. 6 is a flow diagram illustrating an embodiment of a competitivebinding study based on agglutination;

FIG. 7 is a flow diagram illustrating an embodiment of a competitivebinding study based on enzymatic activity;

FIG. 8 is a flow diagram illustrating an embodiment of a competitivebinding study based on chemiluminescence;

FIG. 9 is a schematic diagram illustrating an embodiment of a synthesisprotocol for synthesizing a lamotrigine analog;

FIG. 10 is a schematic diagram illustrating an embodiment of synthesisprotocols for synthesizing lamotrigine analogs;

FIG. 11 is a schematic diagram illustrating an embodiment of a synthesisprotocol for synthesizing a lamotrigine analog;

FIG. 12 is a schematic diagram illustrating an embodiment of a synthesisprotocol for synthesizing a lamotrigine analog;

FIGS. 13A-13E are schematic diagrams illustrating embodiments ofsynthesis protocols for synthesizing a lamotrigine-based immunogens;

FIGS. 14A-14D are schematic diagrams illustrating embodiments ofsynthesis protocols for synthesizing a lamotrigine-based antigens;

FIGS. 15A-15B are schematic diagrams illustrating embodiments ofsynthesis protocols for synthesizing a lamotrigine-based antigens;

FIGS. 16A-16B are schematic diagrams illustrating embodiments ofsynthesis protocols for synthesizing lamotrigine-based fluorescenttracers;

FIG. 17 are schematic diagrams of lamotrigine and embodiments oflamotrigine metabolites;

FIG. 18 is a graph of an embodiment of a comparative study between anautomated immunoassay and an HPLC method; and

FIG. 19 is a graph of a an embodiment of a comparative study between anautomated immunoassay and an HPLC method.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Generally, the present invention relates to lamotrigine analogs andimmunodiagnostic assays for lamotrigine. The lamotrigine analogs caninclude immunogenic moieties that can be used to prepareanti-lamotrigine antibodies, or antigenic moieties or tracer moietiesthat can be used in immunodiagnostic assays for lamotrigine.Additionally, the lamotrigine analogs can be used in immunodiagnosticassays to compete with lamotrigine for anti-lamotrigine antibodies. Assuch, the following terminology is meant to describe embodiments of theinvention, and is not intended to be limiting.

As used herein, the term “hapten” is meant to refer to a partial orincomplete antigen, and can be a small molecule or drug. Also, a haptencan be a low molecular weight molecule that is a protein-free orpolypeptide-free substance. Usually, a hapten is not capable ofstimulating antibody formation alone, but can be capable of interactingwith antibodies. Accordingly, lamotrigine and lamotrigine analogs inaccordance with the present invention can be haptens.

As used herein, the term “analog” or “derivative” is meant to refer to achemical compound or molecule made from a parent compound or molecule byone or more chemical reactions. As such, an analog can be a compoundwith a structure similar to that of lamotrigine or based on alamotrigine scaffold, but differing from it in respect to certaincomponents or structural makeup, which may have a similar or oppositeaction metabolically. An analog or derivative of lamotrigine inaccordance with the present invention can be used to compete for bindingwith an antibody that recognize both the analog and lamotrigine. Also,an analog can include an operative group coupled to lamotrigine througha linker group.

As used herein, the terms “immunogen” and “immunogenic” are meant torefer to substances capable of producing or generating an immuneresponse in an organism. An immunogen can also be antigen. Usually, animmunogen has a fairly high molecular weight (e.g., greater than10,000), thus, a variety of macromolecules such as proteins,lipoproteins, polysaccharides, some nucleic acids, and certain of theteichoic acids, can be coupled to a hapten in order to form an immunogenin accordance with the present invention.

As used herein, the term “immunogenicity” is meant to refer to theability of a molecule to induce an immune response, which is determinedboth by the intrinsic chemical structure of the injected molecule and bywhether or not the host animal can recognize the compound. Small changesin the structure of an antigen can greatly alter the immunogenicity of acompound, and have been used extensively as a general procedure toincrease the chances of raising an antibody, particularly againstwell-conserved antigens. For example, these modification techniqueseither alter regions of the immunogen to provide better sites for T-Cellbinding or expose new epitopes for B-cell binding.

As used herein, the terms “carrier,” “immunogenic moiety,” or“immunogenic carrier,” are meant to refer to an operative group that isan immunogenic substance, commonly a protein, that can be coupled to ahapten. An immunogenic moiety coupled to a hapten can induce an immuneresponse and elicit the production of antibodies that can bindspecifically with the hapten. Immunogenic moieties are operative groupsthat include proteins, polypeptides, glycoproteins, complexpolysaccharides, particles, nucleic acids, polynucleotides, and the likethat are recognized as foreign and thereby elicit an immunologicresponse from the host. Additionally, linkers can comprise modified orunmodified nucleotides, nucleosides, polymers, sugars and othercarbohydrates, polyethers such as, for example, polyethylene glycols,polyalcohols, polypropylenes, propylene glycols, mixtures of ethyleneand propylene glycols, polyalkylamines, polyamines such as spermidine,polyesters such as poly(ethyl acrylate), polyphosphodiesters, andalkylenes. An example of an operative group and its linker ischolesterol-TEG-phosphoramidite, wherein the cholesterol is theoperative group and the tetraethylene glycol and phosphate serve aslinkers.

In one example, an operative group is an immunogenic carrier that can becoupled with a hapten in order to stimulate immunogenicity and antibodyformation against the hapten. Usually, immunogenic carriers are largemolecules that are highly immunogenic and capable of impartingimmunogenicity to a hapten. For example, a protein can be used as animmunogenic carrier because foreign proteins can elicit such animmunological response. Protein carriers can be highly soluble andinclude functional groups that could facilitate easy conjugation with ahapten molecule. Some of the most common carrier proteins in use todayare keyhole limpet hemocyanin (KLH; MW 450,000 to 13,000,000), andbovine serum albumin (BSA, MW 67,000). Keyhole limpet hemocyanin is theoxygen-carrying protein of the marine keyhole limpet, and is extremelylarge and exhibits increased immunogenicity when it is disassociatedinto subunits, probably due to exposure of additional epitopic sites tothe immune system. BSA is highly soluble protein containing numerousfunctional groups suitable for conjugation.

As used herein, the term “antibody” is meant to refer to a protein thatis produced in response to the presence of foreign molecules in thebody. They can be characterized by their ability to bind both toantigens and to specialized cells or proteins of the immune system.Antibodies are divided into five classes, IgG, IgM, IgA, IgE, and IgD,and are immunoglobulin produced by plasma cells.

As used herein, the term “epitope” is meant to defines the region of anantigen that interacts with an antibody. Accordingly, a molecule orother substance, which is an antigen, can include at least one epitopewith antibody activity. This can allow for an antigen to have variousepitopes recognized by the same or different antibody. Also, an epitopeis not an intrinsic property of any particular structure, but can bedefined as a binding site that interacts with the antibody.

As used herein, the term “affinity” is meant to refer to a measure ofthe strength of binding between an epitope and an antibody. Accordingly,a single antibody can have a different affinity for various epitopes.This can allow a single antibody to bind strongly to one epitope andless strongly to another. As such, an antibody can have a first affinityto a drug, such as lamotrigine, and have a second affinity to alamotrigine analog. However, it is possible for the antibody to havesubstantially equivalent or similar affinity for both lamotrigine and alamotrigine analog, which allows the analog to be used to generateantibodies for lamotrigine, and their use in competitive bindingstudies. Thus, lamotrigine analogs in accordance with the presentinvention can be used to generate antibodies with affinity forlamotrigine.

As used herein, the term “avidity” is meant to refer to a measure of theoverall stability of the complex between antibodies and antigens. Theoverall stability of an antibody-antigen interaction can be governed bythree major factors as follows: (a) the intrinsic affinity of theantibody for the epitope; (b) the valency of the antibody and antigen;and (c) the geometric arrangement of the interacting components. Assuch, the avidity of the antibody-antigen complex can be modulated byvarying the foregoing parameters, as well as others.

As used herein, the term “specificity” is meant to refer to thepreferential binding of an antibody with an epitope in comparison withother available epitopes. That is, the specificity of an antibody canpreferentially bind lamotrigine and/or analog instead of a lamotriginemetabolite. This can be used to generate anti-lamotrigine antibodiesthat preferentially bind with lamotrigine over its metabolites so thatthe true concentration of lamotrigine can be assessed so as to not becontaminated by adverse antibody-metabolite binding. Also, thespecificity of an antibody for binding with lamotrigine can be used totailor analogs with similar or substantially the same specificity aslamotrigine.

As used herein, the term “polyclonal antibody” is meant to refer to aheterogeneous mixture of antibodies with a wide range of specificitiesand affinities to a given antigen or epitope. Thus, the polyclonalantibody can include a plurality of antibodies, each distinguishablefrom the others, that bind or otherwise interact with an antigen. Thedifferent antibodies that comprise a polyclonal antibody can be producedor generated by injecting an immunogen having an epitope into an animaland, after an appropriate time, collecting and optionally purifying theblood fraction containing the antibodies of interest. In producingantibodies, several parameters can be considered with respect to thefinal use for the polyclonal antibody. These parameters include thefollowing: (1) the specificity of the antibody (i.e., the ability todistinguish between antigens); (2) the avidity of the antibody (i.e.,the strength of binding an epitope); and (3) the titer of the antibody,which determines the optimal dilution of the antibody in the assaysystem.

As used herein, the term “monoclonal antibody” is meant to refer to anantibody that is isolated from a culture of normal antibody-producingcells and one progenitor cell. A monoclonal antibody can have ahomogeneous binding constant, and are well known in the art.

As used herein, “antibody titer” is meant to refer to the reciprocal ofthe serum dilution. Titers are reported this way for more convenientreporting and formatting. The titer of 1/50000 means that the antibodyeffectively detects the epitope of an antigen when bound together whenthe antigen is at a dilution of 1:50000. The titer is calculated by endpoint titer having about 10% of the maximum O.D.

As used herein, the terms “immunoassay” or “immunodiagnostic” are meantto refer to laboratory techniques that make use of the binding betweenan antigen and an antibody in order to identify and/or quantify at leastone of the specific antigen or specific antibody in a biological sample.Currently, there are three classes of immunoassay, which are describedas follows: (1) antibody capture assays; (2) antigen capture assays; and(3) two-antibody sandwich assays. Additionally, it is contemplated thatnew immunoassays will be developed and will be capable of employing theanalogs and antibodies of the present invention.

As used here, the term “competitive immunoassay” is meant to refer to anexperimental protocol in which a known amount of an identifiable antigencompetes with another antigen for binding with an antibody. That is, aknown antigen that binds with a known antibody is combined with a samplethat is suspected of containing another antigen that also binds with theknown antibody. This allows for the known antigen and another antigen toboth compete for the binding site on the antibody. For example, alamotrigine analog that binds with an anti-lamotrigine antibody can becombined with a sample suspected of containing lamotrigine, and theanalog and lamotrigine compete for binding with the anti-lamotrigineantibody. The competition for binding with the antibody can then be usedto determine whether or not lamotrigine is present in the sample, andcan further be used to quantify the amount of lamotrigine in the sample.

As used herein, the term “turbidimetric detection” is meant to refer tothe measurement of a decrease in the intensity in the transmission, oran increase in absorbance, of incident light due to light scattered byagglutinated particles. A decrease in intensity of transmitted light ismeasured against a higher starting background intensity of transmittedlight. Usually, the reading is made with a detector in line with thelight source, wherein the agglutination of particles inhibitstransmission of the light. Therefore, the inhibition or promotion ofagglutination can be used as a means for assessing the presence of atarget analyte, such as lamotrigine. Turbidimetric assays may be easilyadapted to a variety of clinical analyzers.

As used herein, the term “microparticle agglutination assays” is meantto refer to immunoassays that use the principle of inhibitingagglutination of microparticles by a target analyte. That is, decreasedagglutination is attributed to the presence of the target analyte. Forexample, a derivative of the target drug is covalently linked to thesurface of microparticle and/or the sensitized particles areagglutinated by a monoclonal antibody. When a sample contains free drugthe agglutination is inhibited in proportion to the drug concentration,which leads to a classic inhibition curve relating drug concentration toabsorbance.

As used herein, the term “operative group” is meant to refer to amolecule or macromolecule coupled to lamotrigine through a linker group.An operative group can include an immunogenic moiety, antigen moiety,tracer moiety, and the like. Additionally, the Z group in the chemicalscaffolds described herein is an operative group. As such, the operativegroup can be coupled to the Y linker group and provide an additionalfunctionality to the analong.

As used herein, the terms “active ester” or “activated ester” are meantto refer to an ester group that can react with a free amino group of acompound such as, for example, peptides and proteins. An active estercan include a carboxyl group linked to an active leaving group. Often,the active leaving group includes the ester oxygen so the active leavinggroup removes the ester oxygen. For example, an active ester issusceptible to being displaced by a primary amine, which results in theremoval of the ester oxygen and formation of an amide group. Examples ofactive leaving groups that form active esters includeN-hydroxysuccinimide (“NHS”), p-nitrophenyl, pentafluorophenyl,N-hydroxybenzotriazolyl, and the like. Accordingly, use of the term“NHS” is meant to be defined as N-hydroxysuccinimide.

As used herein, the terms “label,” “detector molecule,” or “tracer” aremeant to refer to any operative group which produces, or can be inducedto produce, a detectable signal. The label can be conjugated tolamotrigine, lamotrigine analog, hapten, analyte, immunogen, antibody,or to another molecule such as a receptor or a molecule that can bind toa receptor. Non-limiting examples of tracers include radioactiveisotopes, enzymes, enzyme fragments, enzyme substrates, enzymeinhibitors, coenzymes, catalysts, fluorophores, dyes, chemiluminescers,luminescers, sensitizers, non-magnetic or magnetic particles, solidsupports, liposomes, ligands, receptors, hapten radioactive isotopes,and the like. As described herein, the analogs can also be coupled to avariety of labels by methods well known in the art to provide a varietyof reagents useful in various immunoassay formats. For detecting theresults of the immunoassays, detector molecules such as fluorophores,for example, fluorescein, radio-labels, or chemiluminescent groups canbe coupled to the analogs to produce tracers.

As used herein, the terms “linking group” or “linker” are meant to referto a portion of a chemical structure that connects two or moresubstructures such as lamotrigine, lamotrigine analogs, haptens, andoperative groups, such as immunogenic moieties, carriers, immunogens,labels, tracers, and the like. A linking group can have at least oneuninterrupted chain of atoms other than hydrogen (or other monovalentatoms) extending between the substructures. Usually, a linking groupincludes a chain of carbon atoms or hetero atoms, which can besubstituted or unsubstituted. The atoms of a linking group and the atomsof a chain within a linking group can be interconnected by chemicalbonds. For example, linkers maybe straight or branched, substituted orunsubstituted, saturated or unsaturated chains, wherein the chain atomscan include carbon and/or hetero atoms. This can include one or morehetero atoms within the chain or at termini of the chains. Additionally,a linking group may also include cyclic and/or aromatic groups as partof the chain or as a substitution on one of the atoms in the chain. Thenumber of atoms in a linking group or linker is determined by countingthe atoms other than hydrogen in the backbone of the chain, which is theshortest route between the substructures being connected. Linking groupsmay be used to provide an available site on a hapten for conjugating ahapten with a tracer, label, carrier, immunogenic moiety, and the like.

As used herein, the term “hetero atoms” is meant to refer to atoms otherthan carbon atoms such as oxygen, nitrogen, sulfur, phosphorus, and thelike. Usually, a heteroatom is multivalent so as to form at least twocovalent bonds, which can be used in a linking group or other moiety.

As used herein, the term “biological sample” is meant to refer to asolid or fluid sample that is obtained from a biological entity. Assuch, a biological sample can include, but is not limited to, anyquantity of a substance from a living thing or formerly living thing,such as humans and other animals. Such a substance can include, but isnot limited to, blood, serum, plasma, urine, tears, cells, organs,tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue,chondrocytes, synovial macrophages, endothelial cells, skin, and thelike.

As used herein, the term “patient” is meant to refer to human and otheranimal subjects. More particularly, a patient is a human or other animalsubject needing an anti-epileptic drug such as lamotrigine.

The lamotrigine analogs can include a lamotrigine molecule coupled to alinker moiety, and optionally include an operative group. The linkermoiety and operative group can be any of a wide range of chemicalcompounds that can modify the physicochemical properties of lamotrigine.Accordingly, the linker moiety can be comprised of an alkyl, aliphatic,straight chain aliphatic, branched aliphatic, substituted aliphatic,cyclic aliphatic, heterocyclic aliphatic, aromatic, heteroaromatic,polyaromatic, and the like.

As used herein, the term “aliphatic” is meant to refer to a hydrocarbylmoiety, such as an alkyl group, that can be straight or branched,saturated or unsaturated, and/or substituted or unsubstituted, which hastwenty or less carbons or hetero atoms in the backbone. Additionally, analiphatic can include 10 or less carbons or hetero atoms in thebackbone. An aliphatic group may comprise moieties that are linear,branched, cyclic and/or heterocyclic, and contain functional groups suchas ethers, ketones, aldehydes, carboxylates, and the like. Exemplaryaliphatic groups include but are not limited to substituted and/orunsubstituted groups of methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, alkylgroups of higher number of carbons and the like, as well as2-methylpropyl, 2-methyl-4-ethylbutyl, 2,4-diethylpropyl, 3-propylbutyl,2,8-dibutyldecyl, 6,6-dimethyloctyl, 6-propyl-6-butyloctyl,2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl, and thelike. The terms aliphatic or alkyl also encompasses alkenyl groups, suchas vinyl, allyl, aralkyl and alkynyl groups.

Substitutions within an aliphatic group can include any atom or groupthat can be tolerated in the aliphatic moiety, including but not limitedto halogens, sulfurs, thiols, thioethers, thioesters, amines (primary,secondary, or tertiary), amides, ethers, esters, alcohols, oxygen, andthe like. The aliphatic groups can by way of example also comprisemodifications such as azo groups, keto groups, aldehyde groups, carbonylgroups, carboxyl groups, nitro, nitroso or nitrile groups, heterocyclessuch as imidazole, hydrazino or hydroxylamino groups, isocyanate orcyanate groups, and sulfur containing groups such as sulfoxide, sulfone,sulfide, and disulfide. Additionally, the substitutions can be viasingle, double, or triple bonds, when relevant or possible.

Further, aliphatic groups may also contain hetero substitutions, whichare substitutions of carbon atoms, by hetero atoms such as, for example,nitrogen, oxygen, phosphorous, or sulfur. As such, a linker comprised ofa substituted aliphatic can have a backbone comprised of carbon,nitrogen, oxygen, sulfur, phosphorous, and/or the like. Heterocyclicsubstitutions refer to alkyl rings having one or more hetero atoms.Examples of heterocyclic moieties include but are not limited tomorpholino, imidazole, and pyrrolidino.

As used herein, the term “aromatic” is meant to refer to molecule is onein which electrons are free to cycle around circular or cyclicarrangements of atoms, which are alternately singly and doubly bonded toone another. More properly, these bonds may be seen as a hybrid of asingle bond and a double bond, each bond in the ring being identical toevery other. Examples of aromatic compounds that can be present inlamotrigine analogs include benzene, benzyl, toluene, xylene, and thelike. The aromatic compound can include hetero atoms so as to be ahetero aromatic such as pyridine, furan, tetrahydrofuran, and the like.Also, an aromatic can be a polycyclic aromatic such as naphthalene,anthracene, phenanthrene, polycyclic aromatic hydrocarbons, indole,quinoline, isoquinoline, and the like.

As used herein, the term “amine” is meant to refer to moieties that canbe derived directly or indirectly from ammonia by replacing one, two, orthree hydrogen atoms by other groups, such as, for example, alkylgroups. Primary amines have the general structures RNH₂ and secondaryamines have the general structure R₂NH. The term amine includes, but isnot limited to methylamine, ethylamine, propylamine, isopropylamine,aniline, cyclohexylamine, benzylamine, polycyclic amines, heteroatomsubstituted aryl and alkylamines, dimethylamine, diethylamine,diisopropylamine, dibutylamine, methylpropylamine, methylhexylamine,methylcyclopropylamine, ethylcylohexylamine, methylbenzylamine,methycyclohexylmethylamine, butylcyclohexylamine, morpholine,thiomorpholine, pyrrolidine, piperidine, 2,6-dimethylpiperidine,piperazine, and heteroatom substituted alkyl or aryl secondary amines.

As used herein, the term “poly(amino acid)” or “polypeptide” is apolyamide formed from amino acids. Poly(amino acid)s will generallyrange from about 200-2,000 molecular weight or greater than about 2,000molecular weight, or having no upper molecular weight limit, andnormally being less than 10,000,000 and usually not more than about600,000 daltons. There will usually be different ranges, depending onwhether an immunogenic carrier or an enzyme is involved.

As used herein, the term “peptide” is meant to refer to any compoundformed by the linkage of two or more amino acids by amide (peptide)bonds, usually a polymer of α-amino acids in which α-amino group of eachamino acid residue (except the NH₂ terminus) is linked to the α-carboxylgroup of the next residue in a linear chain. The terms “peptide,”“polypeptide,” and “poly(amino acid)” are used synonymously herein torefer to this class of compounds without restriction as to size. Thelargest members of this class are referred to as proteins having adefined polypeptide sequence.

Additionally, the terms used herein to describe the invention can beconstrued using the foregoing definitions and/or definitions well knownin the art. As such, the foregoing terminology is meant to describe theinvention and is not intended to be limiting.

I. Lamotrigine Analogs

In one embodiment, the present invention relates to analogs oflamotrigine, which can be used to prepare lamotrigine analog-basedtracers, immunogens and/or analogs. The lamotrigine analogs can beprepared as derivatives of the benzene ring, such as a 4- or 5-positionsubstitution on the benzene ring at the 4 and 5 atoms, and/orderivatives of the triazine ring, such as a 3-position substitution onthe triazine ring at the 3 atom, as shown below. The lamotrigine analogscan be coupled to an immunogenic moiety in order to produce alamotrigine analog-based immunogens that can be used in preparingmonoclonal and polyclonal antibodies. Accordingly, the antibodiesgenerated using unique lamotrigine immunogens can interact and/or bindwith lamotrigine and the analogs. These antibodies, immunogens,antigens, and analogs can be useful in preparing for and performingimmunoassays for the detection of lamotrigine in biological fluids.

In one embodiment, the present invention describes novel analogs oflamotrigine having 5-substitutions at the 5 atom on the benzene ring.That is, the benzene ring can be conjugated to a linking moiety and/oranalog moiety at the 5 atom so as to form an analog. The analog moietycan be considered to be the substituent that is coupled with thelamotrigine scaffold in order to form the analog. The analog moiety canbe any of a wide array of chemical entities, which are described in moredetail below. Accordingly, the 5-substitution analog of lamotrigine canhave the generic structure of Formula 1A and/or Formula 1B:

The lamotrigine scaffold depicted in Formula 1A and/or Formula 1B can besubstituted with a wide range of chemical entities. However, it has beenfound that a 5-substitution succinylamino has unfavorable qualities.Although a 5-succinylamino analog (e.g., succinamic acid) oflamotrigine, which is know as chemical 74W86, can be coupled to acarrier directly, it is highly polar with poor solubility in most commonsolvents. Moreover, activated esters or activated species, which can begenerated in situ, are highly unstable. The coupling between 74W86 andcarrier protein is very inefficient and do not produce anti-lamotrigineantibodies that are sufficient for use in immunoassays, which usuallyhave a low titer. Thus, the following descriptions of the analog moietycan be construed to be exclusive of the 5-succinylamino analog. However,74W86 can be further modified for improved physiochemical properties inorder to produce a lamotrigine analog in accordance with the presentinvention.

Additionally, with respect to Formulas 1A and 1B, when L and W cooperateso as to form an amide bond coupled to the lamotrigine scaffold, W, X,and Y cannot cooperate so as to form HOOC(CH₂)₂C═O. Thus, L-W—X—Y doesnot form a 5-succinylamide group.

In another embodiment, the lamotrigine scaffold can include a4-substitution similar to the 5-substitution. Accordingly, the4-substitution analog of lamotrigine can have the generic structure ofFormula 2A and/or Formula 2B:

With regard to Formulas 2A and 2B, when Z is nothing and L and Wcooperate so as to form an amide bond coupled to the lamotriginescaffold, W, X, and Y can cooperate so as to form HOOC(CH₂)₂C═O. Assuch, the analog moiety can consist of a 4-succinylamido substitution.

In another embodiment, the lamotrigine scaffold can include a3-substitution in the triazine ring. Accordingly, the 3-substitutionanalog of lamotrigine can have the generic structure of Formula 3Aand/or Formula 3B:

With regard to Formulas 3A and 3B, when Z is nothing and L and Wcooperate so as to form an amide bond coupled to the lamotriginescaffold, W, X, and Y can cooperate so as to form HOOC(CH₂)₂C═O. Assuch, the analog moiety can consist of a 4-succinylamido substitution.

The lamotrigine scaffolds depicted in Formulas 1A, 1B, 2A, 2B, 3A, and3B, L can be a wide range of chemical entities. Accordingly, the L groupcan be selected from the group NH (amino), NHCO (amide), SO₂, O, andaliphatic groups. As such, the L group can be used as a linking group toconjugate the analog moiety and/or immunogenic moiety to the lamotriginescaffold.

Additionally, the W group can be an aliphatic, which can be exemplifiedas a saturated or unsaturated, substituted or unsubstituted, andstraight or branched chain having 1-10 carbon or hetero atoms. The Xgroup can be at least one of an aliphatic, bond between W and Y, asubstituted or unsubstituted aromatic or aliphatic group having from 1-2rings, and/or a saturated or unsaturated, substituted or unsubstituted,or straight or branched chain having 1-10 carbon or hetero chain atoms.Some examples of substitutions on the aliphatic linker groups includeprimary and secondary amines, carbonyl groups, halogens, and the like.

The Y group can be an end group or a coupling group, which can be usedfor coupling the linker group with an operative group, such as acarrier, label, immunogenic moiety, and the like. Also, the Y group canbe a reactive group that is used to couple the linking group to the Zgroup. As such, Y can be various groups, such as aliphatics, amines,amides, carboxylic acids, aldehydes, esters, activated esters, aliphaticesters, imidoesters, isocyanates, isothiocyanates, anhydrides, thiols,alcohols, thiolactones, diazoniums, maleimido groups, and the like.Also, Y can be a Y₁—Z, wherein Y₁ is linking group derived from the Yend group or coupling group being coupled to the Z group.

Furthermore, the operative group Z can be nothing or any moiety that canbe coupled to the linker moiety. As such, the L-W—X—Y group can beconsidered to be the linker moiety and the Z group can be an operativegroup. As such, the linker moiety can functionally serve as a linker orlinking group between the lamotrigine scaffold and an operative group.For example, the operative group can be a carrier, label, tracer,protein, enzyme, fluorogenic compound, phosphorogenic compound,thermochromic compound, photochromic compound, anti-stokes up-regulatingcompound, chemiluminescent material, electrochemical mediator, particle,reporter group, enzyme inhibitor, nucleic acid, polypeptide, and thelike.

For example, the W group can comprise a chain of one or more atoms,wherein at least one atom is carbon if present. Illustratively, W can beany of the following groups: CH₂; (CH₂)₂; (CH₂)₃; (CH₂)₄; (CH₂)₅;(CH₂)₆; CH₂CO; (CH₂)₂CO; (CH₂)₃CO; (CH₂)₄CO; (CH₂)₅CO; (CH₂)₆CO; CH₂COO;(CH₂)₂COO; (CH₂)₃COO; (CH₂)₄COO; (CH₂)₅COO; (CH₂)₆COO; CO; COO; COCH₂;CO(CH₂)₂; CO(CH₂)₃; CO(CH₂)₄; CO(CH₂)₅; CO(CH₂)₆; COCH₂CO; CO(CH₂)₂CO;CO(CH₂)₃CO; CO(CH₂)₄CO; CO(CH₂)₅CO; CO(CH₂)₆CO; COCH₂COO; CO(CH₂)₂COO;CO(CH₂)₃COO; CO(CH₂)₄COO; CO(CH₂)₅COO; CO(CH₂)₆COO; CO(CH₂)₂CONHCH₂;CO(CH₂)₂CONH(CH₂)₂; CONH(CH₂)₃; CONH(CH₂)₃CO; CONH(CH₂)₃COO; NHCH₂;NH(CH₂)₂; NH(CH₂)₃; NH(CH₂)₄; NH(CH₂)₅; NH(CH₂)₆; NHCH₂CO; NH(CH₂)₂CO;NH(CH₂)₃CO; NH(CH₂)₄CO; NH(CH₂)₅CO; NH(CH₂)₆CO; NHCH₂COO; NH(CH₂)₂COO;NH(CH₂)₃COO; NH(CH₂)₄COO; NH(CH₂)₅COO; NH(CH₂)₆COO; NHCO(CH₂)₂;NHCO(CH₂)₆; NHCO(CH₂)₂CO; HCO(CH₂)₆CO; NHCO(CH₂)₂COO; or NHCO(CH₂)₆COO;combinations thereof; and the like. More preferably, W is selected fromthe group consisting of CH₂j (CH₂)₂, (CH₂)₃, CH₂COO, (CH₂)₂CO,(CH₂)₂COO, (CH₂)₃CO, (CH₂)₃COO, CO(CH₂)₆, CO(CH₂)₆CO, CO(CH₂)₆COO, CO,COO CONH(CH₂)₃, CONH(CH₂)₃CO, CONH(CH₂)₃COO, CO(CH₂)₂, COCH₂,CO(CH₂)₂CONHCH₂, CO(CH₂)₂CONH(CH₂)₂, combinations thereof, and the like.Most preferably, W is selected from the group consisting of CO(CH₂)₂,COCH₂, CO(CH₂)₂CONHCH₂, and CO(CH₂)₂CONH(CH₂)₂. In one embodiment, W canbe an aliphatic group having from 5 to 10 carbon and/or hetero chainatoms.

For example, the X group can be a bond or a chain of zero or more atoms,wherein at least one atom is carbon if present. As such, X can be acovalent bond between L and Y. Illustratively, X can be any of thefollowing groups: CH₂; (CH₂)₂; (CH₂)₃; (CH₂)₄; (CH₂)₅; (CH₂)₆; CH₂CO;(CH₂)₂CO; (CH₂)₃CO; (CH₂)₄CO; (CH₂)₅CO; (CH₂)₆CO; CH₂COO; (CH₂)₂COO;(CH₂)₃COO; (CH₂)₄COO; (CH₂)₅COO; (CH₂)₆COO; CO; COO; COCH₂; CO(CH₂)₂;CO(CH₂)₃; CO(CH₂)₄; CO(CH₂)₅; CO(CH₂)₆; COCH₂CO; CO(CH₂)₂CO; CO(CH₂)₃CO;CO(CH₂)₄CO; CO(CH₂)₅CO; CO(CH₂)₆CO; COCH₂COO; CO(CH₂)₂COO; CO(CH₂)₃COO;CO(CH₂)₄COO; CO(CH₂)₅COO; CO(CH₂)₆COO; CO(CH₂)₂CONHCH₂;CO(CH₂)₂CONH(CH₂)₂; Ph; CONHCH₂Ph; CONH(CH₂)₃; CONH(CH₂)₃CO;CONH(CH₂)₃COO; NHCH₂; NH(CH₂)₂; NH(CH₂)₃; NH(CH₂)₄; NH(CH₂)₅; NH(CH₂)₆;NHCH₂CO; NH(CH₂)₂CO; NH(CH₂)₃CO; NH(CH₂)₄CO; NH(CH₂)₅CO; NH(CH₂)₆CO;NHCH₂COO; NH(CH₂)₂COO; NH(CH₂)₃COO; NH(CH₂)₄COO; NH(CH₂)₅COO;NH(CH₂)₆COO; NHCO(CH₂)₂; NHCO(CH₂)₆; NHCO(CH₂)₂CO; HCO(CH₂)₆CO;NHCO(CH₂)₂COO; or NHCO(CH₂)₆COO; combinations thereof; and the like.More preferably, X is selected from the group consisting of CH₂, (CH₂)₂,(CH₂)₃, CH₂COO, (CH₂)₂CO, (CH₂)₂COO, (CH₂)₃CO, (CH₂)₃COO, CO(CH₂)₆,CO(CH₂)₆CO, CO(CH₂)₆COO, CO, COO, Ph, CONH(CH₂)₃, CONH(CH₂)₃CO,CONH(CH₂)₃COO, combinations thereof, and the like. Most preferably, X isselected from the group consisting of CH₂, CONHCH₂Ph, CONH(CH₂)₂, Ph,NHCO(CH₂)₆, and NHCO(CH₂)₂.

For example, in each of Formulas 1 and 2 the Y group can comprise an endgroup or linker derived from the end group and is always present.Illustratively, Y can be any of the following end groups or a linkergroup derived therefrom: COOH (carboxylic acid); COO; COO—NHS(NHS activeester); NHS; COO-tertbutyl; tertbutyl (t-butyl); OH; O—NHS(NHS activeester linker); COOCH₂CH₃; COOCH₃; OCH₂CH₃; OCH₃; NH; NH₂; NHCO (amide);combinations thereof; and the like. More preferably, when Y is an endgroup, it is selected from the group consisting of NHS, COOH, COO—NHS,COO-tertbutyl, tertbutyl, OH, O—NHS, COOCH₂CH₃, COOCH₃, OCH₂CH₃, OCH₃,or NH₂. On the other hand, when Y is a linker, it is Y₁—Z, wherein Y₁ isselected from the group consisting of is at least one of COO, CO, O,CONH, or NH and Z is a macromolecule.

Accordingly, the conjugate Z or macromolecule can be a carrier, tracer,or a label, such as protein, enzyme, fluorescent compound,chemiluminescent material, electrochemical mediator, particle, reportergroup, enzyme inhibitor, and/or nucleic acid. Illustratively, Z can beany of the following conjugate groups: (a) BSA; (b) KLH; (c) fluorescenttracer; and (d) the like.

In one embodiment, the lamotragine analog can have L-W—X—Y selected fromthe group consisting of NHCO(CH₂)₂CONH(CH₂)₂NHCOOH,NHCO(CH₂)₂CONH(CH₂)₂NHCOONHS, NHCO(CH₂)₂CONH(CH₂)₂NHCOOCH₂CH₃,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₂COOH,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₂COONHS,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₂COOCH₂CH₃,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₃COOH,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₃COONHS,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₃COOCH₂CH₃,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₆COOH,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₆COONHS,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₆COOCH₂CH₃,NHCO(CH₂)₂CONH(CH₂)₂NHCH₂PhCOOH, NHCO(CH₂)₂CONH(CH₂)₂NHCH₂PhCOONHS,NHCO(CH₂)₂CONH(CH₂)₂NHCH₂PhCOOCH₂CH₃,NHCO(CH₂)₂CONH(CH₂)₂NHCONH(CH₂)₃COOH,NHCO(CH₂)₂CONH(CH₂)₂NHCONH(CH₂)₃COONHS,NHCO(CH₂)₂CONH(CH₂)₂NHCONH(CH₂)₃COOCH₃, NHCO(CH₂)₂CONHCH₂PhCOOH,NHCO(CH₂)₂CONHCH₂PhCOOCH₂CH₃, NHCO(CH₂)₂COOH, NHCO(CH₂)₂COONHS,NHCO(CH₂)₂COOCH₂CH₃, NHCO(CH₂)₃COOH, NHCO(CH₂)₃COONHS,NHCO(CH₂)₃COOCH₂CH₃, NH(CH₂)₂NHCO(CH₂)₆COOH, NH(CH₂)₂NHCO(CH₂)₆COONHS,NH(CH₂)₂NHCO(CH₂)₆COOCH₂CH₃, NH(CH₂)₂NH(CH₂)₃COOC(CH₃)₃,NH(CH₂)₂NH(CH₂)₃COOH, NH(CH₂)₂NH(CH₂)₃COONHS, NHCH₂PhCOOH,NHCH₂PhCOONHS, NHCOPhCOOH, NHCOPhCOONHS, OOCNH(CH₂)₃COOCH₂CH₃,OOCNH(CH₂)₃COOCH₃, OOCNH(CH₂)₃COONHS, OOCNH(CH₂)₃COOH, NH(CH₂)₃COOH,NH(CH₂)₃COONHS, and the like.

In one embodiment, the lamotragine analog can have L-W—X—Y—Z selectedfrom the group consisting of NHCO(CH₂)₂CONH(CH₂)₂NHCOO-BSA,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₂COO-BSA,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₃COO-BSA,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₆COO-BSA,NHCO(CH₂)₂CONH(CH₂)₂NHCH₂PhCOO-BSA,NHCO(CH₂)₂CONH(CH₂)₂NHCONH(CH₂)₃COO-BSA, NHCO(CH₂)₂CONHCH₂PhCOO-BSA,NHCO(CH₂)₂COO-BSA, NHCO(CH₂)₃COO-BSA, NH(CH₂)₂NHCO(CH₂)₆COO-BSA,NH(CH₂)₂NH(CH₂)₃COO-BSA, NHCH₂PhCOO-BSA, NHCOPhCOO-BSA,OOCNH(CH₂)₃COO-BSA, NH(CH₂)₃COO-BSA, and the like.

In one embodiment, the lamotrigine analogs of Formulas 1A, 2A, and 3Acan be used as therapeutic agents. As such, the lamotrigine analogs canbe used as anti-epileptic drugs similarly as lamotrigine. However, whena lamotrigine analog is used as an therapeutic agent, Z is preferablynothing so as to not form an immunogen. Thus, the non-immunogenicanalogs of lamotrigine can be used in anti-epileptic regimens foranimals, including humans.

II. Lamotrigine Immunogens

Implementing an immunoassay for the detection of a small molecule, suchas lamotrigine, can be a challenge. This is because such small moleculescan often lack antigenicity, which makes it difficult to generateantibodies against lamotrigine, and is particularly problematic withlamotrigine, which lacks immunogenicity. To increase the immunogenicity,larger antigenic compounds, including but not limited to bovine serumalbumin, ovalbumin, keyhole limpet hemocyanin, and the like, can becoupled to the drug. Further, detection of the drug in an immunoassaygenerally requires the use of a detectable label conjugated to anantibody, lamotrigine, or lamotrigine analog.

Immunogens may be made by coupling lamotrigine to an antigenic carrierprotein through a linker of one of the lamotrigine analogues. As such,an immunogen based on lamotrigine is also considered a lamotrigineanalog. Illustratively, a 5-amino substitution on the benzene ring hasbeen shown to link with a protein, which is described by Sailstad et al,Ther Drug Monitoring 13:433-442 (1991), which is incorporated herein byreference. However, the antibodies generated from the immunogendescribed by Sailstad et al. were not satisfactory in any immunoassay,especially automated immunoassays. The poor immunogenicity can beattributed to poor titer, poor sensitivity, BSA not being as immunogenicas KLH, and a short linker at the 5-position.

In an attempt to improve the efficiency of coupling to carrier protein,74W85 was modified with NHS into an activated ester derivative5-NHS-74W86 (5) isolated (e.g., L, NH; W is C═O; X is CH₂CH₂; Y is NHSactivated ester), as shown in FIG. 10. The 5-NHS active ester (5) can beefficiently coupled to a carrier protein because it reacts directly withlysine or other amines within the protein. Examples of such a couplingcan be seen in the immunogen 5-KLH-74W86 (20) of FIG. 13E. However, thelinker of (20) proved to be too short to provide an accessible epitopefor antibody interaction in two immunizations programs described in moredetail below.

Due to the unsuccessful immunization programs, new haptens forlamotrigine were explored extensively, which included longer linkersconjugated to the 5-position, and linkers of various length conjugatedto the 4-position and 3-position. Accordingly, FIGS. 13A-13D illustrateimmunogens prepared in accordance with the present invention and are asfollows: (a) 3-lamotrigine immunogen (18); (b) 5-long linker lamotrigineimmunogen (16); (c) 5-long linker lamotrigine immunogen (19); and (d)4-lamotrigine immunogen (17). Specifically, 5-long linker lamotrigineimmunogens (16) and (19) have much longer linkers to provide for a moreaccessible epitope. As such, the lamotrigine moiety is much moreaccessible for the antibody interaction and is much more immunogenic.

In one embodiment, the present invention relates to immunogens preparedfrom the forgoing lamotrigine analog scaffolds. Namely, the analogs ofFormulas 2B, 3B, and 4B can include the linker moieties as describedabove, and Z can be an operative group, such as an immunogenic moiety.As such, Z can be any immunogenic moiety that can elicit animmunological response and provide for antibodies to be produced thattarget at least a portion of the lamotrigine analog.

An immunogenic moiety can include various proteins or polypeptides,which can function as an immunogenic carrier. These types ofpolypeptides include albumins, serum proteins, globulins, ocular lensproteins, lipoproteins, and portions thereof. Illustrative proteinsinclude bovine serum albumin (“BSA”), keyhole limpet hemocyanin (“KLH”),egg ovalbumin, bovine gamma-globulin (“BGG”), and the like.Alternatively, synthetic polypeptides may be utilized. Additionally, animmunogenic moiety can also be a polysaccharide, which is a highmolecular weight polymer. Examples of polysaccharides are starches,glycogen, cellulose, carbohydrate gums such as gum arabic, agar, and thelike. Also, an immunogenic moiety can be a polynucleotide, such as DNAor RNA. The polynucleotide can be modified or unmodified, and becomprised of any number of nucleic acids so long as it provides thecarrier and/or immunogenic functionality. The polysaccharide can alsocontain or link to a polypeptide residue, polynucleotide residue, and/orlipid residues. Furthermore, an immunogenic moiety can also be apolynucleotide either alone or conjugate to one of the polypeptides orpolysaccharides mentioned above.

An immunogenic moiety or carrier can also be a particle ormicroparticle. The immunogenic particles are generally at least about0.02 microns (urn) and not more than about 100 μm, and usually about0.05 μm to 10 μm in diameter. The particle can be organic or inorganic,swellable or non-swellable, and/or porous or non-porous. Optionally, animmunogenic particle can have a density approximating water, generallyfrom about 0.5 to 1.5 g/ml, and be composed of a material that can betransparent, partially transparent, or opaque. The immunogenic particlescan be biological materials such as cells and microorganisms, includingnon-limiting examples such as erythrocytes, leukocytes, lymphocytes,Streptococcus, Staphylococcus aureus, E. coli, and viruses. Theparticles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, liposomes, cationic liposomes,anionic liposomes, lipoproteins, lipopolymers, and the like.

In one embodiment, the lamotragine analog can have L-W—X—Y—Z selectedfrom the group consisting of NHCO(CH₂)₂CONH(CH₂)₂NHCOO-KLH,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₂COO-KLH,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₃COO-KLH,NHCO(CH₂)₂CONH(CH₂)₂NHCO(CH₂)₆COO-KLH,NHCO(CH₂)₂CONH(CH₂)₂NHCH₂PhCOO-KLH,NHCO(CH₂)₂CONH(CH₂)₂NHCONH(CH₂)₃COO-KLH, NHCO(CH₂)₂CONHCH₂PhCOO-KLH,NHCO(CH₂)₂COO-KLH, NHCO(CH₂)₃COO-KLH, NH(CH₂)₂NHCO(CH₂)₆COO-KLH,NH(CH₂)₂NH(CH₂)₃COO-KLH, NHCH₂PhCOO-KLH, NHCOPhCOO-KLH,OOCNH(CH₂)₃COO-KLH, NH(CH₂)₃COO-KLH, and the like.

Thus, the immunogens prepared in accordance with the present inventioncan be used to generate antibodies that can have an affinity forlamotrigine as well as lamotrigine analogs.

III. Antibodies for Lamotrigine and Lamotrigine Analogs

In one embodiment, a lamotrigine analog-based immunogen in accordancewith the present invention can be used in an embodiment of a method forproducing monoclonal and/or polyclonal antibodies. As such, antibodiescan be produced from the lamotrigine-based immunogen that interactsand/or binds with lamotrigine. This can allow for the analogs of thepresent invention to be useful in preparing antibodies for use inimmunoassays for identifying the presence of lamotrigine. Also, methodsof producing antibodies with immunogens are well known in the art. Theimmunogens can be used in the screening for the monoclonal and/orpolyclonal antibodies that interact and/or bind with lamotrigine.

Additionally, the sera can be obtained, processed and/or purified bywell-known techniques for collecting antibodies. As such, monoclonaland/or polyclonal antibodies can be obtained that interact and/or bindwith both lamotrigine and a lamotrigine analog. This allows for thelamotrigine-immunogen to be used in preparing antibodies that recognizelamotrigine and can be used in immunodiagnostic assays.

FIG. 1 is a flow diagram illustrating one embodiment of a method 10 forobtaining anti-lamotrigine antibodies, an immunogen based on alamotrigine analog can be obtained (Block 12). The immunogen can then becombined with an immunogenic formulation (Block 14). Briefly, about 0.5of an immunogen composition is admixed with about 0.5 ml of completeFreund's adjuvant; however, other amounts of immunogen and/or adjuvantcan be used. The immunogenic formulation can then be administered to anantibody producing subject (Block 16), which can be a rat, mouse, pig,rabbit, bird and/or other animal, but preferably mammals. Theadministration can be via tail vein injection, subcutaneous injection,intravenous injection, or other well-known injection sites.Subsequently, immunogenic boosters can be administered to the animalthat received the initial administration (Block 18), wherein the boostercan include substantially the same ingredients as the initialformulation and can be administered at predetermined intervals. Forexample, the initial administration can be followed by subsequentboosters once a week or at other longer or shorter intervals. After atleast the initial administration, and optionally after subsequentboosters, the anti-lamotrigine antibodies produced by the animal can becollected (Block 20). The antibodies can be collected by obtainingblood, serum, plasma, or other biological sample from the animalpreviously administered the immunogen. Optionally, heantibody-containing composition can then be processed as is well knownin the art (Block 22), wherein such processing can include techniquesthat place the antibodies into a format suitable for performing animmunodiagnostic assay. Alternatively, the processing can includescreening the antibodies with ELISA by well known and establishedtechniques. As such, the processing can be used to obtain polyclonalantibodies (Block 24), which can also result in purifying polyclonalantibodies (Block 26). Alternatively, techniques well known in the artcan be used to obtain monoclonal antibodies, which can also result inpurifying monoclonal antibodies.

IV. Immunodiagnostic Assays

The anti-lamotrigine antibodies, either monoclonal or polyclonal, can beused in immunoassays for identifying the presence of lamotrigine in asample, such as blood, plasma, serum, tissue, and the like. This can bebeneficial for identifying or accessing pharmacokinetic and/orpharmacodynamic parameters for lamotrigine in a patient or patientpopulation. Thus, the anti-lamotrigine antibodies can be used inimmunodiagnostic assays in place of other antibodies so that the assayscan be configured for identifying the presence and optionally,quantifying the amount of lamotrigine. Additionally, theimmunodiagnostic assays can use lamotrigine analogs in accordance withthe present invention or other lamotrigine analogs.

A. Fluorescence Polarization Immunoassay for Lamotrigine

Fluorescence polarization immunoassay (FPIA) technology is based uponcompetitive binding between an antigen/drug in a sample and a knownconcentration of labeled antigen/drug. FPIA technology is described inU.S. Pat. Nos. 4,593,089, 4,492,762, 4,668,640, and 4,751,190, which areincorporated herein by reference. Accordingly, the FPIA reagents,systems, and equipment described in the incorporated references can beused with anti-lamotrigine antibodies which are also anti-lamotrigineanalog antibodies.

The FPIA technology can be used to identify the presence of lamotrigineand can be used in assays that quantify the amount of lamotrigine in asample. In part, the rotational properties of molecules in solutionallow for the degree of polarization to be directly proportional to thesize of the molecule. Accordingly, polarization increases as molecularsize increases. That is, when linearly polarized light is used to excitea fluorescent-labeled or other luminescent-labeled lamotrigine or analogthereof, which is small and rotates rapidly in solution, the emittedlight is significantly depolarized. When the fluorescent-labeledlamotrigine or analog interacts with or is bound to an antibody, therotation is slowed and the emitted light is highly polarized. This isbecause the antibody significantly and measurably increases the size ofthe complex. Also, increasing the amount of unlabeled lamotrigine in thesample can result in decreased binding of the fluorescent-labeledlamotrigine or analog by the anti-lamotrigine antibody, and therebydecrease the polarization of light emitted from sample. The quantitativerelationship between polarization and concentration of the unlabeledlamotrigine in the sample can be established by measuring thepolarization values of calibrations with known concentrations oflamotrigine. Thus, FPIA can be used to identify the presence andconcentration of lamotrigine in a sample.

One embodiment of the present invention is an FPIA assay system. Anexample of components of the FPIA system can include the following: i)monoclonal or polyclonal anti-lamotrigine antibodies capable of bindingto lamotrigine and a lamotrigine analog; ii) a sample suspected ofcontaining the lamotrigine; and iii) lamotrigine analog labeled with afluorescent moiety, such as fluorescein. Alternatively, the system canbe provided as a kit exclusive of the sample. Additionally, the systemcan include various buffer compositions, lamotrigine concentrationgradient compositions or a stock composition of lamotrigine, and thelike.

FIG. 2 is a flow diagram illustrating one embodiment of a method 110 forperforming a FPIA assay. As such, a luminescent-labeled lamotrigine oranalog conjugate can be obtained (Block 112), and an anti-lamotrigineantibody can be obtained (Block 114). Additionally, a sample, such as abiological sample from a patient being administered lamotrigine,suspected of containing lamotrigine can be obtained (Block 116). Knownamounts or concentrations of luminescent-labeled lamotrigine conjugateand anti-lamotrigine antibody can be obtained and formulated intoseparate compositions, such as in a standard buffer system, for use in acompetitive binding assay (Block 118). The anti-lamotrigine antibody andluminescent-labeled lamotrigine conjugate are then combined with thebiological sample into a reaction solution (Block 120). A competitivereaction takes place between the luminescent-labeled lamotrigineconjugate and the unknown amount of lamotrigine in the biological samplewith the anti-lamotrigine antibody in the reaction solution (Block 122).After adequate duration and/or competition the luminescent conjugate isilluminated (Block 124), which can be by photoillumination,chemical-illumination, temperature-illumination, and the like. Thepolarization of the light emitted by the illumination is then measured(Block 126) and compared to polarization values of known amounts oflamotrigine and/or luminescent conjugate (Block 128), which can be usedto determine whether or not lamotrigine is present in the sample (Block130). Additionally, comparing the measurements obtained from thebiological sample with standardized measurements obtained from knownconcentration standards can be used to quantify the amount oflamotrigine in the sample (Block 132), and thereby identify the amountof lamotrigine in the patient (Block 134).

B. Homogeneous Microparticle Immunoassay for Lamotrigine

Homogeneous microparticles immunoassay (“HMI”) technology, which can bereferred to as immunoturbidimetric assays, is based on the agglutinationof particles and compounds in solution. When particles and/or chemicalcompounds agglutinate, particle sizes can increase and increase theturbidity of a solution. Accordingly, anti-lamotrigine antibodies can beused with microparticles and lamotrigine analogs in order to assess thepresence, and optionally the amount, of lamotrigine in a sample. HMItechnologies can be advantageous because the immunoassays can beperformed on blood, blood hemolysate, serum, plasma, tissue, and/orother samples. HMI assays can be configured to be performed withlamotrigine and/or an analog loaded onto a microparticle, or with ananti-lamotrigine antibody loaded onto a microparticle. The use of ananalog loaded microparticle can be especially advantageous because ofthe ability to efficiently load the microparticle. In any event, HMI orimmunoturbidimetric assays are well known in the art for measuringagglutination of substances in a sample.

Immunoturbidimetric assay technologies are described in U.S. Pat. Nos.5,571,728, 4,847,209, 6,514,770, 6,248,597, which are included herein byreference. Briefly, in homogeneous assay methods use is madepredominantly of light attenuation, nephelometric, or turbidimetricmethods. The formation of an agglutinated compound AB from lamotrigine(A) and anti-lamotrigine antibody microparticle binding partner (B) canbe measured by the change which occurs in the scattering or absorptionof the incident light directed into the sample. Alternatively, theanti-lamotrigine antibody (A) can bind with a lamotrigine or analogloaded microparticle. When suspendable particles having an immobilizedbinding partner are used, there is an enhancement of the effects, whichmakes it possible to determine considerably lower lamotrigineconcentrations. These homogeneous methods can be carried out quickly andsimply, and permit, in particular, the automation of sample analyses asdescribed in more detail below.

For example, in high volume screening applications it can be desirableto have fully automated methods of analysis. As such, instruments can bedesigned to detect changes in light scattering by particles, such assensitized latex particles, as a result of specific reaction withanalyte. The assays that utilize such instruments can be made highlysensitive due to the vast surface area of latex particle suspensions andthe physical principles of light scattering. The main principle ofdetection involves the light scattering change when two or moreparticles come into close contact during agglutination. When a beam oflight is passed through a reaction cell containing un-agglutinatedparticles, there can be a certain degree of light scatter due torefraction, reflection, absorption, and diffraction by the particles.Accordingly, this principle can be beneficial for measuring the abilityof a target analyte, such as lamotrigine to inhibit agglutination ofparticles. During the early stages of an antibody/antigen binding,complexes begin to form, wherein these complexes can substantially alterthe angular distribution of the scattered light intensity because thecomplexes act like larger particles. The change of light scatter as aresult of larger particles by agglutination may be measured byturbidimetric detection and other methods, as described in more detailbelow. Seradyn's Lamotrigine QMS® reagents permit the completeautomation and are applicable to many clinical chemistry analyzers.

FIG. 3 is an illustration of a competition assay that combines anantibody buffer with a biological sample having a free drug, such aslamotrigine, and a hapten coated particle reagent, wherein the haptencan be a lamotrigine analog. In the instance the biological samplecontains little or no lamotrigine, there is no inhibition ofagglutination. As the amount of lamotrigine in the sample increases,there can be partial inhibition so as to result in only partialagglutination. Additionally, a large amount of lamotrigine in the samplecan result in the complete inhibition of agglutination. Thus, theanalysis of agglutination can be used to identify the presence oflamotrigine. Also, the use of a standardized curve of lamotrigineconcentrations, as shown in FIG. 4, can be used to identify the amountof lamotrigine in the sample based on the absorbance change fromagglutination.

i. Lamotrigine Loaded Microparticles

FIG. 5 is a flow diagram illustrating one embodiment of a method 210 forperforming an HMI assay. Accordingly, lamotrigine analogs can beobtained (Block 212) and loaded on a microparticle (Block 214), such asany of the microparticles manufactured and/or sold by Seradyn, Inc.(Indianapolis, Ind.), which can include polystyrene,carboxylate-modified polystyrene, streptavidin-coated magneticparticles, and the like. A sample, such as a biological sample from apatient being administered lamotrigine, suspected of containinglamotrigine can be obtained (Block 216). An anti-lamotrigine antibody,such as monoclonal or polyclonal, capable of specifically bindinglamotrigine and lamotrigine analogs in accordance with the presentinvention is obtained (Block 218), and then optionally formulated in astandard buffer system (Block 220). The antibody composition is thencombined with the lamotrigine-microparticle and biological sample (Block222), wherein the amounts of antibody and lamotrigine analog bound tothe microparticle are known. A competitive reaction takes place betweenlamotrigine analog immobilized on the microparticles and the lamotriginein the biological sample for binding to a limited amount ofanti-lamotrigine antibody in the reaction solution (Block 224).Agglutination of lamotrigine-loaded microparticles with antibody isinhibited by the presence of lamotrigine in the biological sample,wherein agglutination inhibition is directly proportional toconcentration of lamotrigine in the biological sample. This allows forthe presence of lamotrigine in the sample to be determined by well-knownturbidimetric assays (Block 226). Additionally, comparing themeasurements obtained from the biological sample with standardizedmeasurements obtained from known concentration standards can be used toquantify the amount of lamotrigine in the sample (Block 228), andthereby identify the amount of lamotrigine in the patient (Block 230).

One embodiment of the present invention is a lamotrigine loadedmicroparticle HMI assay system. An example of components of the HMIsystem can include the following: i) monoclonal or polyclonalanti-lamotrigine antibodies capable of binding lamotrigine and alamotrigine analog; ii) a sample suspected of containing thelamotrigine; and iii) lamotrigine analog coupled to a microparticle,such as a polystyrene microparticle. Alternatively, the system can beprovided as a kit without the sample. Additionally, the system caninclude various buffer compositions, lamotrigine concentration gradientcompositions or a stock composition of lamotrigine, and the like.

ii. Anti-Lamotrigine Loaded Microparticles

In another embodiment, which is similar to that described above withrespect to lamotrigine loaded microparticles, an anti-lamotrigineantibody capable of binding lamotrigine and a lamotrigine analog isloaded on the microparticle. The lamotrigine analog can include anoperative group of choice, for example, bovine serum albumin, ovalbumin,dextran, and the like. A competitive reaction takes place between thelamotrigine analog and lamotrigine in the patient's sample for bindingto anti-lamotrigine antibody immobilized on the microparticles. Again,agglutination of particles is inhibited by the presence of drug inpatient sample.

FIG. 6 is a flow diagram illustrating another embodiment of a method 310for performing an HMI assay. Accordingly, anti-lamotrigine antibodiescapable of specifically binding lamotrigine and a lamotrigine analog canbe obtained (Block 312) and loaded on a microparticle (Block 314). Asample, such as a biological sample from a patient being administeredlamotrigine, suspected of containing lamotrigine can be obtained (Block316). A lamotrigine analog can be obtained, where the analog can includea suitable operative group (Block 318). Known amounts or concentrationsof the lamotrigine analog and anti-lamotrigine antibody-loadedmicroparticle are then formulated into separate compositions, such as astandard buffer system, for use in a competitive binding assay (Block320). The antibody-microparticle composition is then combined with thelamotrigine analog composition and biological sample (Block 322). Acompetitive reaction takes place between the lamotrigine analog andlamotrigine in the biological sample with the anti-lamotrigine antibodyimmobilized on the microparticles in the reaction solution (Block 324).Agglutination of anti-lamotrigine antibody-loaded microparticles withthe lamotrigine analog is inhibited by the presence of lamotrigine inthe biological sample, wherein inhibition of agglutination is directlyproportional to concentration of lamotrigine in the biological sample.This allows for the presence of lamotrigine in the sample to bedetermined by well-known turbidimetric assays (Block 326). Additionally,comparing the measurements obtained from the biological sample withstandardized measurements obtained from known concentration standardscan be used to quantify the amount of lamotrigine in the sample (Block328), and thereby identify the amount of lamotrigine in the patient(Block 330)

One embodiment of the present invention is an anti-lamotrigine antibodyloaded microparticle HMI assay system. An example of components of theHMI system can include the following: i) microparticles loaded withmonoclonal or polyclonal anti-lamotrigine antibodies that are capable ofbinding to lamotrigine and a lamotrigine analog; ii) a sample suspectedof containing the lamotrigine; and iii) a lamotrigine analog, which canoptionally include a macromolecule or other carrier. Alternatively, theassay system can be provided exclusive of the sample, which can beprovided later or from another source. Additionally, the assay systemcan include various buffer compositions, lamotrigine concentrationgradient compositions or a stock composition of lamotrigine or analog,and the like.

C. Cloned Enzyme Donor Immunoassays for Lamotrigine

Cloned enzyme donor Immunoassays (“CEDIA®” a trademark of RocheDiagnostics) has proven to be a highly accurate and effective method foridentifying the presence and performing quantitative measurements oftherapeutic drugs. The CEDIA® technology has been described in detail inthe following patents: (a) U.S. Pat. No. 4,708,929 disclosingcompetitive homogeneous assay methods; (b) U.S. Pat. No. 5,120,653disclosing a recombinant DNA sequence for coding the enzyme donorfragment and a host for such a vector; (c) U.S. Pat. No. 5,604,091disclosing amino acid sequences of the enzyme donor fragment; and (d)U.S. Pat. No. 5,643,734 which teaches kits for CEDIA assays, wherein allof the foregoing patents are incorporated herein by reference. Briefly,CEDIA® technology is based upon the competition of a lamotrigine in thebiological sample with analog conjugated to an inactive geneticallyengineered enzyme-donor (“ED”) fragment such as from β-D-galactosidegalactohydrolase or β-galactosidase (“β gal”) from E. coli, for bindingto an antibody capable of specifically binding lamotrigine. In theinstance the lamotrigine is present in the sample it binds to theantibody, leaving the ED portion of the ED-analog conjugate free torestore enzyme activity of β-D-galactoside galactohydrolase or β gal inthe reaction mixture so as to be capable of association with enzymeacceptor (“EA”) fragments. The active enzyme is then capable ofproducing a quantifiable reaction product when exposed to an appropriatesubstrate. A preferred substrate is chlorophenolred-β-D-galactopyranoside (“CPRG”), which can be cleaved by the activeenzyme having the ED and EA fragments into galactose and CPR, whereinCPR is measured by absorbency at about wavelength 570 nm. In theinstance lamotrigine is not present in the sample, the antibody binds tothe ED-analog conjugate, thereby inhibiting association of the EDfragments with the EA fragments and inhibiting restoration of enzymeactivity. The amount of reaction product and resultant absorbance changeare proportional to the amount of lamotrigine in the sample.

FIG. 7 is a flow diagram illustrating one embodiment of a method 410 forperforming a CEDIA® assay. Accordingly, a lamotrigine-ED conjugate canbe obtained (Block 412), which can be by conjugating a lamotrigineanalog with the ED. Also, an EA corresponding with the ED can beobtained (Block 414). Additionally, a sample, such as a biologicalsample from a patient being administered lamotrigine, suspected ofcontaining lamotrigine can be obtained (Block 416). Anti-lamotrigineantibody, which can also interact with the lamotrigine-ED conjugate canbe obtained by methods in accordance with the present invention (Block418). Known amounts or concentrations of lamotrigine-ED conjugate, EA,and anti-lamotrigine antibody are obtained and formulated into separatecompositions, such as a standard buffer system, for use in a competitivebinding assay (Block 420). The lamotrigine-ED conjugate and antibody isthen combined with the biological sample into a reaction solution (Block422). Optionally, the EA is also combined into the reaction solution atthis point or later after a sufficient time for competitive interactionswith the antibody to occur. A competitive reaction takes place betweenthe lamotrigine-ED conjugate and lamotrigine in the biological samplewith the anti-lamotrigine antibody in the reaction solution (Block 424).After the competitive reactions and the EA has been introduced into thereaction solution, an ED-EA enzyme-cleavable substrate is introducedinto the reaction solution (Block 426). The enzyme activity between theED-EA enzyme and enzyme-cleavable substrate is measured (Block 428),which can be by measuring the absorbance of a cleavage product or otherwell-known measuring technique. The measurement of enzyme activity canbe used to determine whether or not lamotrigine is present in the sample(Block 430). Additionally, comparing the measurements obtained from thebiological sample with standardized measurements obtained from knownconcentration standards can be used to quantify the amount oflamotrigine in the sample (Block 432), and thereby identify the amountof lamotrigine in the patient (Block 434).

One embodiment of the present invention is a CEDIA® assay system. Anexample of components of the CEDIA® system can include the following: i)monoclonal or polyclonal anti-lamotrigine antibodies capable of bindingto lamotrigine, a lamotrigine analog, and/or lamotrigine-ED orlamotrigine-EA; ii) a sample suspected of containing the lamotrigine;iii) a lamotrigine analog coupled to an ED or EA; and iv) one of an EDor EA that will associate with the lamotrigine-ED or lamotrigine-EA forrestoring enzymatic activity so that an ED and EA are present in thesystem. Alternatively, the assay system can be provided as a kitexclusive of the sample. Additionally, the assay system can includevarious buffer compositions, lamotrigine concentration gradientcompositions or a stock composition of lamotrigine, and the like.

D. Chemiluminescent Heterogeneous Immunoassays for Lamotrigine

A competitive assay using chemiluminescent microparticle immunoassay(“CMIA”) technology can also be used to assess whether or notlamotrigine is present in a sample. Various types of CMIA technologiesare well known in the art of heterogeneous immunoassays for determiningthe presence and/or amount of a chemical entity in a sample, whereinsome CMIA technologies can be exemplified by U.S. Pat. Nos. 6,448,091,5,798,083, and 5,834,206, which are incorporated herein by reference.Such CMIA assays can include the use of anti-lamotrigine antibodies,which are capable of specifically binding to lamotrigine and it analogs,coupled to particles, such as particular magnetic particles or particlessuitable for separation by filtration, sedimentation, and/or othermeans. Additionally, a tracer, which can include a lamotrigine analoglinked to a suitable chemiluminescent moiety, for example an acridiniumester, can be used to compete with free lamotrigine in the patient'ssample for the limited amount of anti-lamotrigine antibody on theparticle. After the sample, tracer, and antibody particles interact anda routine wash step has removed unbound tracer, the amount of tracerbound to antibody particles can be measured by chemiluminescence,wherein chemiluminescence is expressed in Relative Light Units (RULE).The amount of chemiluminescence is inversely related to the amount offree drug in the patient's sample and concentration is determined byconstructing a standard curve using known values of the drug.

FIG. 8 is a flow diagram illustrating one embodiment of a method 510 forperforming a CMIA assay. Accordingly, an anti-lamotrigineantibody-particle conjugate can be obtained (Block 512), which can beperformed by coupling the antibody with a particle such as a magneticparticle. Also, a tracer compound including a lamotrigine analog havinga chemiluminescent moiety can be obtained (Block 514). Additionally, asample, such as a biological sample from a patient being administeredlamotrigine, suspected of containing lamotrigine can be obtained (Block516). Known amounts or concentrations of tracer and anti-lamotrigineantibody-particle conjugate can be formulated into separatecompositions, such as a standard buffer system, for use in a competitivebinding assay (Block 518). The anti-lamotrigine antibody-particleconjugate and tracer is then combined with the biological sample into areaction solution (Block 520). A competitive reaction takes placebetween the tracer and lamotrigine in the biological sample with theanti-lamotrigine antibody-particle conjugate in the reaction solution(Block 522). After sufficient duration and/or binding competition, theantibody-particle conjugate is separated from the reaction solution(Block 524). Optionally, any unbound lamotrigine and/or tracer can beremoved from the antibody-particle conjugate by a wash or otherseparation technique (Block 526). The amount of chemiluminescence can bedetermined by exciting the tracer so that the chemiluminescent moietyemits light by phosphorescence, fluorescence, or other luminescencewhich is measurable (Block 528). Often, the chemiluminescence isfluorescence, which is measured in RLUs. The measurement ofchemiluminescence can be used to determine whether or not lamotrigine ispresent in the sample (Block 530). Additionally, comparing measurementsobtained from the biological sample with standardized measurementsobtained from known concentration standards can be used to quantify theamount of lamotrigine in the sample (Block 532), and thereby identifythe amount of lamotrigine in the patient (Block 534).

One embodiment of the present invention is a CMIA assay system. Anexample of components of the CMIA system can include the following: i)particles or microparticles loaded with monoclonal or polyclonalanti-lamotrigine antibodies that are capable of binding to lamotrigineand a lamotrigine analog; ii) a sample suspected of containing thelamotrigine; and iii) an analog tracer. Alternatively, the assay systemcan be provided as a kit exclusive of the sample. Additionally, thesystem can include various buffer compositions, lamotrigineconcentration gradient compositions or a stock composition oflamotrigine or analog, and the like.

E. Other Immunoassays for Lamotrigine

The lamotrigine analogs, conjugates, antibodies, immunogens and/or otherconjugates described herein are also suitable for any of a number ofother heterogeneous immunoassays with a range of detection systemsincluding but not limited to enzymatic or fluorescent, and/orhomogeneous immunoassays including but not limited to rapid lateral flowassays, and antibody arrays, as well as formats yet to be developed.

While various immunodiagnostic assays have been described herein thatutilize the lamotrigine analogs, conjugates, antibodies, immunogensand/or tracers, such assays can also be modified as is well known in theart. As such, various modifications of steps or acts for performing suchimmunoassays can be made within the scope of the present invention.

EXAMPLES

The following examples are provided to illustrate embodiments of theprevention and are not intended to be limiting. Accordingly, some of theexamples have been performed via experiment and some are prophetic basedon techniques, standards, and results well known in the art. Also, itshould be apparent that the invention can include additional embodimentsnot illustrated by example. Additionally, many of the examples have beenperformed with experimental protocols well known in the art using thelamotrigine analogs, antigens, immunogens, and anti-lamotrigineantibodies prepared in accordance with the present invention. Thus, theexamples can be supplemented with the following references, which areall incorporated herein by reference: (a) Caryl Griffin et al.,Microparticle Reagent Optimization: A Laboratory Reference Manual fromthe Authority on Microparticles, Seradyn (1994); and (b) BoehringerMannheim Corporation Technical Publications Department, HitachiOperation Manual Version B, Boehringer Mannheim Corporation LaboratoryDiagnostic Division (1992); and (c) the NCCLS, approved guideline Aug.2004.

Example 1

FIG. 9 is a schematic representation of a chemical reaction forconverting lamotrigine (1) into 5-intermediates and a 5-succinylaminolamotrigine derivative (“74W86”) (4), which can be further synthesizedinto various lamotrigine analogs in accordance with the presentinvention. Accordingly, lamotrigine (1) is treated with a mixture ofnitric acid and sulfuric acid to form 5-nitro lamotrigine analog (2).The 5-nitro lamotrigine analog (2) is then isolated and reduced withhydrogen and Pd catalyst to form the resulting 5-amino lamotrigineanalog (3), which is acylated with succinic anhydride to form the5-succinylamino derivative of lamotrigine, (“74W86”) (4). The5-succinylamino derivative of lamotrigine, (“74W86”) (4) is thenpurified.

Example 2

FIG. 10 is a schematic representation of a chemical reaction forconverting 74W86 (4) into an NHS-active ester form in order to improvethe efficiency of coupling to carrier proteins and the like.Accordingly, 74W86 (4), obtained via the chemistry described in Example1, is modified with NHS to obtain the activated ester derivative oflamotrigine, which is 5-NHS-74W86 (5). The active ester 5-NHS-74W86 (5)can be efficiently coupled to a carrier protein or other moiety since itreacts directly with the amine of lysine within the protein, and otheramine groups. Also, 5-NHS-74W86 (5) can be coupled to linking groupsthat have already been conjugated to a carrier protein, wherein thelinker includes an amine group to form an amide linking group.

Specifically, a solution of 745 mg of 74W86 (4) in 25 mL anhydrous DMFis cooled to 0° C., and 0.7 mL N,N-diisopropylethyl amine is added toform a reaction mixture. The reaction mixture is reacted by the additionof 785 mg of O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate. The reaction mixture is allowed to warm up to roomtemperature and stirred overnight. The reaction mixture is concentratedunder reduced pressure, and the residue is purified by flash columnchromatography using ethyl acetate/methanol as eluent to giveapproximately 500 mg of active ester derivative 5-NHS-74W86 (5).

Example 3

With continuing reference to FIG. 10, the 5-NHS-74W86 (5) lamotrigineanalog, which is obtained via the chemical reaction of Example 2, isconjugated with a linking group. More specifically, the 5-NHS-74W86 (5)analog is reacted with an alkyldiamine, such as ethylenediamine, toproduce a lamotrigine analog 5-ethylenediamine 74W86 (6). The reactionis performed by adding 0.5 mL of ethylenediamine to a solution of 300 mgof 5-NHS-74W86 (5) in 3 mL anhydrous DMF to form a reaction mixture. Thereaction mixture is stirred at room temperature overnight andconcentrated until dry under reduced pressure. The dried residue ispurified by flash column chromatography using methanol/ammoniumhydroxide as eluent to give approximately 200 mg lamotrigine analog5-ethylenediamine 74W86 (6). The 5-ethylenediamine 74W86 (6) can beactivated for conjugation with additional linking groups or reacteddirectly with carboxyl groups on other molecules.

Example 4

With continuing reference to FIG. 10, the 5-ethylenediamine 74W86 (6)lamotrigine analog, which is obtained via the chemical reaction ofExample 3, is further conjugated with a linking group. Morespecifically, 5-ethylenediamine 74W86 (6) is acylated by being reactedwith succinic anhydride to form a 5-lamtorigine analog (7). The reactionis conducted with about 0.2 mL N,N-diisopropylethyl amine being added toa suspension of 75 mg of lamotrigine derivative (6) in 5 mL anhydrousDMF to form a reaction mixture. The reaction mixture is stirred for 5min followed by the addition of 83 mg of succinic anhydride. Thereaction mixture is stirred for 2 h and concentrated until dry underreduced pressure. The dry residue is purified by flash columnchromatography using methanol/ethyl acetate as eluent to giveapproximately 200 mg 5-lamotrigine analog (7).

Example 5

With continuing reference to FIG. 10, the 5-ethylenediamine 74W86 (6)lamotrigine analog, which is obtained via the chemical reaction ofExample 3, is further conjugated with a linking group. Morespecifically, 5-ethylenediamine 74W86 (6) is acylated by reaction withdisuccinimidyl suberate (“DSS”) to form the 5-lamotrigine analog (8).The reaction is conducted with about 0.2 mL N,N-diisopropylethyl aminebeing added to a solution of 2 g of DSS in 6 mL DMF that is chilled inan ice bath, and followed by addition of a suspension of 327 mg of5-ethylenediamine 74W86 (6) in 15 mL anhydrous DMF to form a reactionmixture. The reaction mixture is stirred for 4 h and concentrated untildry under reduced pressure. The residue is purified by flash columnchromatography using methanol/ethyl acetate as eluent to giveapproximately 350 mg 5-lamotrigine analog (8).

Example 6

With continuing reference to FIG. 10, the 5-NHS-74W86 (5) lamotrigineanalog, is further conjugated with a linking group. Accordingly, in around bottom flask containing a magnetic stirrer, about 59 mg of5-NHS-74W86 (5) and 40 mg of methyl 4-aminomethyl benzoate hydrochlorideare combined. About 2 mL of anhydrous DMF and 0.1 ml ofN,N-diisopropylethylamine are added to the flask, and stirred under Arin a 60° C. oil bath. The reaction is stopped after 24 h. The volatilesare evaporated under reduced pressure and the residue is purified byflash column chromatography using ethyl acetate/methanol as eluent togive 20 mg lamotrigine analog (9).

Example 7

FIG. 11 is a schematic representation of a chemical reaction forconverting lamotrigine into 4-intermediates and 4-succinylaminolamotrigine. Accordingly, lamotrigine is treated with a mixture ofnitric acid and sulfuric acid to form 4-nitro lamotrigine (10). The4-nitro lamotrigine (10) intermediate is isolated and reduced withhydrogen via a Pd catalyst to form a 4-amino lamotrigine (11)intermediate, which is then acylated with succinic anhydride to form the4-succinylamino lamotrigine (12) analog. The 4-succinylamino lamotrigine(12) analog is purified, and then can be further reacted with linkergroups or carrier moieties in order to form analogs and conjugates inaccordance with the present invention.

Example 8

With continuing reference to FIG. 11, the 4-succinylamino lamotrigine(12) analog is reacted with NHS to form an activated ester such as4-NHS-succinylamino lamotrigine (13) analog. Accordingly, the4-succinylamino lamotrigine (12) analog is reacted with NHS underreaction conditions substantially similar as in Example 2. The4-NHS-succinylamino lamotrigine (13) analog is then purified.

Example 9

FIG. 12 is a schematic representation of a chemical reaction forconverting lamotrigine into 3-intermediates and 3-succinyl lamotrigine(14). Accordingly, about 1.0 mL N,N-diisopropylethyl amine is added to asolution of 2 g of lamotrigine in 30 mL anhydrous DMF. The mixture isstirred for 5 min followed by the addition of 600 mg of succinicanhydride to form a reaction mixture. The reaction mixture is stirredovernight and concentrated until dry under reduced pressure. The dryresidue is purified by flash column chromatography usingmethanol/ammonium hydroxide as eluent to yield approximately 1 g of3-succinyl lamotrigine (14), which is characterized by Formula 4A withthe following: L is NH.

Example 10

With continuing reference to FIG. 12, the 3-succinyl lamotrigine (14) ismodified to an active ester. Accordingly, a solution of 400 mg of3-succinyl lamotrigine (14) in 20 mL anhydrous DMF is cooled to 0° C.,and 0.3 mL N,N-diisopropylethyl amine is added to form a reactionmixture. Subsequently, about 450 mg ofO-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate isadded to the reaction mixture. The reaction mixture is allowed to warmup to room temperature and stirred for 4 h. The reaction mixture is thenconcentrated to obtain a dry residue under reduced pressure, and the dryresidue is purified by flash column chromatography using ethylacetate/methanol as eluent to yield approximately 235 mg of3-NHS-succinyl lamotrigine (14).

Example 11

FIGS. 13A-13D are schematic representations of chemical reactions forconverting lamotrigine analogs to lamotrigine analog conjugates, whichcan be used as immunogens to produce anti-lamotrigine antibodies and toproduce conjugates for use in various immunodiagnostic assays asdescribed herein. More particularly, active esters of lamotrigineanalogs (7), (13), (15) and (8) can be coupled to immunogenic carrierproteins, such as keyhole limpet hemocyanin (“KLH”). FIG. 13A is anschematic diagram illustrating an exemplary synthesis method used toform immunogen (16), which is a 5-KLH-lamotrigine analog conjugatehaving a long linker. The reaction is conducted by cooling a solution of80 mg of KLH in 8 ml PBS (0.1 M sodium phosphate, 0.15 M sodium chloridepH 7.2) in an ice bath. Next, a solution of 18 mg of 5-lamotrigineanalog (7) in 1 mL PBS buffer PH 7.2 is added to the protein solutiondrop-wise to form a reaction mixture. The reaction mixture is allowed tostir at room temperature for 10 minutes then 60 mg ED AC[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] in 0.5 mLDI water is added and stirred for 30 minutes. The resulting conjugate isplaced in a dialysis tube (10,000 MW cut-off), and dialyzed PBS in pH7.2 at 4° C., which is then followed by five changes with PBS at pH 7.2(1 L each for at least 6 hours each). The protein concentration of theresulting immunogen (16) is determined using a BCA assay.

Example 12

FIG. 13B is a schematic diagram illustrating an alternative chemicalreaction to provide an additional immunogen (17). A solution of 60 mg ofKLH in 6 mL PBS at pH 7.2 (0.1 M sodium phosphate, 0.15 M sodiumchloride) is cooled in an ice bath before 3.8 mL of DMSO is added to theKLH solution drop-wise, and maintained below room temperature. Asolution of 17.4 mg of lamotrigine analog (13) in 1 mL DMSO is added tothe protein solution drop-wise to form a reaction mixture. The reactionmixture is allowed to stir at room temperature for 40 h. The resultingconjugate, 4-KLH immunogen (17), is placed in a dialysis tube (10,000 MWcut-off) and dialyzed in serial dialysis baths of 1 L of 35% DMSO in pH7.2 PBS, 1 L of 10% DMSO in pH 7.2 PBS, and 1 L of 10% DMSO in PBS atroom temperature, which is then followed by four changes with PBS at 4°C. (1 L each for at least 6 hours each). The immunogen (17) can be usedin preparing monoclonal and polyclonal antibodies that can interact andbind with lamotrigine and lamotrigine analogs by methods describedherein and well known in the art.

Example 13

FIG. 13C is a schematic diagram illustrating an alternative chemicalreaction to provide an additional immunogen (18). As such, a solution of60 mg of KLH in 6 mL PBS at pH 7.2 (0.1 M sodium phosphate, 0.15 Msodium chloride) is cooled in an ice bath before 3.8 mL of DMSO is addedto the KLH solution drop-wise, and maintained below room temperature. Asolution of 12.7 mg of lamotrigine analog (15) in 1 mL DMSO is added tothe protein solution drop-wise to form a reaction mixture. The reactionmixture is allowed to stir at room temperature for 40 h. The resultingconjugate is placed in a dialysis tube (10,000 MW cut-off) and seriallydialyzed in 1 L of 35% DMSO in PBS at pH 7.2, 1 L of 10% DMSO in PBS atpH 7.2, 1 L of 10% DMSO in PBS at pH 7.2 at room temperature, which isfollowed by four changes with PBS at pH 7.2 at 4° C. (1 L each for atleast 6 hours each). The protein concentration of immunogen (18) isdetermined as approximately 2.17 mg/mL using BCA assay. The resultingimmunogen (18), and other immunogens prepared with similar chemicalreactions, can be used to produce monoclonal and/or polyclonalantibodies by methods described herein and well known in the art.

Example 14

FIG. 13D is a schematic diagram illustrating an alternative chemicalreaction to provide an additional immunogen (19). Accordingly, areaction scheme substantially similar to the reactions described inExamples 11-13 can be employed with lamotrigine analog (8) as thestarting material. Briefly, a solution of 60 mg of KLH in 6 mL PBS at pH7.2 (0.1 M sodium phosphate, 0.15 M sodium chloride) is cooled in an icebath, and 3.8 mL of DMSO are added to the KLH solution drop-wise andmaintained below room temperature. A solution of 17.4 mg of lamotriginederivative (8) in 1 mL DMSO is added to the protein solution drop-wiseto form a reaction mixture. The reaction mixture is stirred at roomtemperature for 40 h. The resulting conjugate, 5-KLH analog (19), isplaced in a dialysis tube (10,000 MW cut-off) and dialyzed in 1 L of 35%DMSO in PBS at pH 7.2, 1 L of 10% DMSO in PBS at pH 7.2, 1 L of 10% DMSOin PBS at pH 7.2 at room temperature, and followed by four changes withPBS at pH 7.2 at 4° C. (1 L each for at least 6 hours each). Theresulting 5-immunogen (19), and other immunogens prepared with similarchemical reactions, can be used to produce monoclonal and/or polyclonalantibodies by methods described herein and well known in the art.

Example 15

Additionally, FIG. 13E is a schematic diagram illustrating a similarreaction scheme to those described in Examples 10-13 can be employedwith lamotrigine analog (5) to make a corresponding immunogen (20).However, it has been determined that the immunogen (19) has a shortlinker at the 5-position, which has resulted in data that shows to yieldpoor results possibly due to the polar nature of 74W86-immunogens.

Example 16

FIG. 14A is a schematic representation of a chemical reaction forconverting a lamotrigine analog to a lamotrigine analog conjugate, whichcan be used as antigens, competitors, and immunogens for producinganti-lamotrigine antibodies in various immunodiagnostic assays asdescribed herein. Accordingly, a reaction scheme substantially similarto the reactions described in Examples 11-14 can be employed withlamotrigine analog (8) as the starting material. Briefly, a solution of60 mg of BSA in 6 mL PBS at pH 7.2 (0.1 M sodium phosphate, 0.15 Msodium chloride) is cooled in an ice bath, and 3.8 mL of DMSO are addedto the KLH solution drop-wise and maintained below room temperature. Asolution of 18.2 mg of lamotrigine derivative (8) in 1 mL DMSO is addedto the protein solution drop-wise to form a reaction mixture. Thereaction mixture is stirred at room temperature for 40 h. The resultingconjugate, 5-BSA analog (19), is placed in a dialysis tube (10,000 MWcut-off) and serially dialyzed in 1 L of 35% DMSO in PBS at pH 7.2, 1 Lof 10% DMSO in PBS at pH 7.2, 1 L of 10% DMSO in PBS at pH 7.2 at roomtemperature, and followed by four changes with PBS at pH 7.2 at 4° C. (1L each for at least 6 hours each). The resulting 5-BSA conjugate (21),can be used as “competitors” in heterogeneous and homogeneousimmunodiagnostic assays as described herein, as well as in ELISAscreening and other immunoassays. Also, immunogens (21) can be used forpreparing anti-lamotrigine antibodies in accordance with the presentinvention, especially when the BSA moiety is substituted with a KLHmoiety.

Example 17

FIG. 14B is a schematic representation of chemical reaction forconverting the 4-NHS-succinylamino lamotrigine analog (13) to an antigenthat can interact with an anti-lamotrigine antibody. Accordingly, the4-lamotrigine analog (13) can be reacted with carrier protein, such asBSA or HSA, in order to form a 4-BSA conjugate or antigen (22), asshown. The synthesis protocol can be substantially similar to thereaction described in Example 16.

Example 18

FIG. 14C is a schematic representation of chemical reaction forconverting the 3-NHS-succinyl lamotrigine analog (5) to a 3-BSAconjugate or antigen (23), as shown. The synthesis protocol can besubstantially similar to the reaction described in Example 16.

Example 19

FIG. 14D is a schematic representation of chemical reaction forconverting the 5-NHS-succinyl lamotrigine analog (14) to a 5-BSAconjugate or antigen (24), as shown. The synthesis protocol can besubstantially similar to the reaction described in Examples 16, 17, and18. However, it has been determined that the antigen (24) has a shortlinker at the 5-position, which has resulted in data that shows to yieldpoor results possibly due to the polar nature of 74W86-antigens.

Example 20

FIG. 15A is a schematic representation of a chemical reaction forconverting a lamotrigine analog to a lamotrigine analog antigen (25).Carboxylate groups (aspartic acid, glutamic acid) in proteins may bederivatized through the use of amide bond forming agents or throughreactive carbonyl intermediates. Accordingly, a carrier protein such asBSA or HSA can be activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (“EDAC”), as described in more detail below,in order to form an active intermediate of the actived BSA or HSA. Theactive intermediate can then be coupled to lamotrigine analog (6), inorder to form a 5-BSA conjugate or antigen (25). The reaction isconducted by cooling a solution of 51 mg of BSA in 4 mL PBS (0.1 pH 7.2M sodium phosphate, 0.15 M sodium chloride) in an ice bath and thesolution is allowed to stir in an ice bath for 30 min. Subsequently, 1mL of DMSO and 10 mg of lamotrigine analog (6) in 0.4 ml DMSO is addeddropwise to the BSA solution. The solution is allowed to warm to roomtemperature while stirring. A solution of 25 mg of EDAC in 0.4 mL DIwater is added to the above BSA protein solution. The solution isstirred for 4 hours. The resulting conjugate is placed in a dialysistube (10,000 MW cut-off) and serially dialyzed in 1 L of 30% DMSO in PBSat pH 7.2, 1 L of 10% DMSO in PBS at pH 7.2, 1 L of 10% DMSO in PBS atpH 7.2 at room temperature, and followed by four changes with PBS at pH7.2 at 4° C. (1 L each for at least 6 hours each). The proteinconcentration of the resulting immunogen (25) is determined using BCAassay to be about 6.4 mg/ml.

Example 21

FIG. 15B is a schematic representation of chemical reactions forconverting lamotrigine analog (7) to lamotrigine analog antigen (26).Accordingly, lamotrigine analog (7) can be coupled to carrier protein insitu via ED AC activation. The resulting immunogens or conjugates (26)can be used as competitor in heterogeneous and homogeneousimmunodiagnostic assays as described herein, as well as in ELISAscreening and other immunoassays. Also, immunogens (26) can be used forpreparing anti-lamotrigine antibodies in accordance with the presentinvention, especially when the BSA moiety is substituted with a KLHmoiety. Accordingly, a 5-lamotrigine analog (7) can be reacted withEDAC, as described in more detail below, in order to form an activeintermediate of the 5-lamotrigine analog (7). The active intermediatecan then be coupled to a carrier protein, such as BSA or HSA, in orderto form a 5-BSA conjugate or antigen (26), as shown. The reaction isconducted by cooling a solution of 51 mg of BSA in 5 mL PBS (0.1 pH 7.2M sodium phosphate, 0.15 M sodium chloride) in an ice bath and thesolution is maintained below room temperature. Subsequently, a solutionof 12 mg of 5-lamotrigine analog (7) in 1 mL PBS pH 7.2 buffer is addedto the protein solution drop-wise. The solution is stirred for 10 minthen is added 40 mg EDAC in 0.5 mL DI water. The reaction mixture isallowed to stir at room temperature for 30 minutes. The resultingconjugate is placed in a dialysis tube (10,000 MW cut-off), and dialyzedin 1 L×7 of 100% PBS in pH 7.2 at 4° C. at room temperature, which isthen followed by five changes with PBS at pH 7.2 (1 L each for at least6 hours each). The protein concentration of the resulting conjugate (26)is determined using BCA assay to be about 6.9 mg/ml.

FIG. 22

FIG. 16A is a schematic diagram illustrating a chemical reaction forcoupling a lamotrigine analog (2) with a fluorescent label, such asFITC. In a round bottom flask wrapped with aluminum foil, a reactionsolution of 10 mg FITC (Fluoresceine-5-isothiocyanate), 0.1 mlN,N-diisopropylethylamine and 8 mg of lamotrigine analog (2) is formed.The reaction solution is stirred for 18 hours, and the volatiles areevaporated under reduced pressure. The residue is re-dissolved inmethanol and purified from preparative TLC plates using solvent ethylacetate/methanol. The tracer (27) is dissolved in methanol and stored infreezer.

Example 23

FIG. 16B is a schematic diagram illustrating a chemical reaction forcoupling a lamotrigine analog (6) with a fluorescent label, such as FAM.In a round bottom flask wrapped with aluminum foil, a reaction solutionof 10 mg FAM (Carboxyfluorescein succinimidyl ester), 0.1 mLN,N-diisopropylethylamine and 12 mg of lamotrigine analog (6) isprepared. The reaction solution is stirred for 18 hours, and thevolatiles are evaporated under reduced pressure. The residue isre-dissolved in methanol and purified from preparative TLC plates usingsolvent ethyl acetate/methanol. The tracer (28) is dissolved in methanoland stored in freezer.

Example 24

A polyclonal antisera is obtained and an assay is performed in order todetermine the amount of cross-reactivity of the polyclonal antibody withlamotrigine and lamotrigine metabolites. A known amount of lamotrigineis used to react with an anti-lamotrigine antibody prepared inaccordance with the present invention. The known concentration oflamotrigine is used to calculate the amount of cross-reactivity betweenthe antibody preparation and the metabolites as follows: N-methyl (29);N-oxide (30); and N-2 gluronide (31). The chemical structures oflamotrigine (1) N-methyl (29); N-oxide (30); and N-2 gluronide (31) areshown in FIG. 17. The percent of cross-reactivity equals 100 times theobserved concentration of lamotrigine in μg/mL, which is then divided bythe concentration of added metabolites in μg/mL. No cross-reactivity isobserved in specimens containing those metabolites. High concentrationsof these compounds are spiked into human serum and tested as samples.The metabolites are assayed and compared to control serum (nolamotrigine). Cross reactivity is calculated using the followingequation:

${\%{\mspace{11mu}\;}{cross}\mspace{14mu}{reactivity}} = {\frac{{Recovered}\mspace{14mu}{Concentration}}{{Concentration}\mspace{14mu}{of}\mspace{14mu}{cross}\mspace{14mu}{reactant}} \times 100\%}$

The results indicate that at a concentration of 500 μg/mL, N-2 methyland N-2 glucuronide do not have any cross-reactivity. Also, thecross-reactivity of N-2 oxide at 500 μg/mL is less then 3%.

Example 25

A polyclonal antibody that binds with lamotrigine is prepared using alamotrigine analog having an immunogenic conjugate. More particularly,the lamotrigine immunogen (20) having the KLH immunogenic moiety wasused to generate the anti-lamotrigine polyclonal antibody, which is wellknown in the art. An immunogenic composition is prepared by mixing about0.5 mL of an immunogen (20) containing composition with about 0.5 mL ofFreund's adjuvant. The resulting 1 mL immunogenic cocktail is theninjected in each rabbit. Subsequent immunogenic injections having thesame cocktail are administered to the rabbits every four weeks in orderto cause the rabbit to produce anti-lamotrigine polyclonal antibody.Sera from two rabbits, Rabbit number 1309 and 1310, are screened viaELISA using antigens, as described below.

Example 26

ELISA plates for use in an ELISA assay were prepared in order to studythe polyclonal antibody prepared as described in Example 25. As such,various lamotrigine antigens (21), (23), and (24) were coated ondifferent ELISA plates before being subjected to the anti-lamotriginepolyclonal antibody and competing free lamotrigine. More particularly,the lamotrigine antigens were diluted in coating buffer, and then addedto the wells of ELISA plate. After the ELISA plate was incubated for 60min at 37° C., the solvent in the coating buffer was decanted and ablocking buffer was added to the plate. The plate was incubated againfor 60 min at 37° C., and the solvent in the blocking buffer wasdecanted from the plate. The ELISA plate was then stored with theblocking agent in the wells at 2-8° C. for up to 1 week.

Example 27

The antibody titer for a polyclonal antibody prepared in accordance withExample 25 was determined using ELISA plates as prepared in Example 26.As such, a serial dilution was performed to produce the same 100 μLvolume in each well. The antibody dilutions were prepared between 1:10and 1:2000 in PBS at pH 7.4 and containing 0.1% BSA. The samples werediluted 10 fold, and the dilutions were started at 1:100 and seriallydiluted 10 fold across the plate. Subsequently, a 100 μL of antibodysample was added to each well on the ELISA plate. The plate was thenincubated for 60 min at 37° C., and washed three times with 250 μL ofPBS at pH 7.4 with 0.05% tween. Next, 125 μL of a diluted secondantibody conjugate (in PBS, pH 7.4), which is different from the antigenprevious coated onto the plate, was added to each well of the plate.Titer was determined experimentally by incubating the plate for 60 minat 37° C., which was then washed three times with 250 μL of PBS at pH7.4 with 0.05% tween. After washing, about 125 μL of2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (“ABTS”) substratewas added to each well in the plates, and the plate was incubated againfor 20 min. The plate was read at 405 nm, and the titer results areprovided in Table 1.

TABLE 1 ELISA Titer Rabbit No. Immunogen Antigen 24 Antigen 23 Antigen21 1309 20 1:12000 1:19000 1:11000 1310 20 1:210  1:1200  1:900 

These results indicate that the titer was not sufficient for themicroparticle agglutination immunoassay. This is because themicroparticles agglutination immunoassay should be conducted with atiter of at least 1:100,000. As such, the immunogen (20) did not producesufficient antibodies for use in commercial immunodiagnostic assayprotocols.

Example 28

The avidity of the anti-lamotrigine antibodies prepared with immunogen(20) for lamotrigine analogs were determined by a binding inhibitionstudy. As such, samples were prepared in 1 mL of PBS at pH 7.4 with 0.1%BSA. A composition having 30% Bmax titer or 50% Bmax titer was used todivide the obtained titer value into approximately half the titer value.Accordingly, this can accommodate a 1:1 dilution which occurs when theanti-lamotrigine antibody is mixed with the inhibiting protein. Using30% Bmax, an antibody titer of 1:12000 is diluted to 1:6000 during thesample preparation stage. About 50 μL of lamotrigine at differentconcentrations or calibrator values, (0, 2.5, 5, 10, 20, 40 μg/ml) werethen applied to the plate as prepared in accordance with Example 25.About 50 μL of the diluted antibody was dispensed into the plate, andcompositions in the plate were mixed for 1 min on a horizontal plateshaker. The plate was characterized by a first row not containinglamotrigine or anti-lamotrigine antibody, and the first row was used asthe negative control. A second row not containing lamotrigine was usedas the positive control. The plate was incubated for 60 min, and washedthree times with 250 μL of PBS at pH 7.4 with 0.05% tween. About 125 μLof a diluted second antibody conjugate, which is different fromimmunogen 15, such as antigens (21), (23), or (24), in PBS at pH 7.4 wasadded to each well of the plate. Titer was determined experimentally bythe plate being incubated for 60 min at 37° C. and washed 3 times with250 μL PBS, pH 7.4 with 0.05% tween. Subsequently, about 125 μL of ABTSsubstrate was added to each well of the plates and the plate wasincubated for 20 min. The plate was read at 405 nm, and the results areprovided in Tables 2 and 3.

TABLE 2 Rabbit No. 1309 Antigen 24 Antigen 23 Antigen 21 Lamotrigine(μg/ml) A₄₀₅ B/Bo A₄₀₅ B/Bo A₄₀₅ B/Bo 0 0.8 1.00 0.4 1.00 0.55 1.00 2.50.78 0.98 0.35 0.88 0.46 0.84 5 0.75 0.94 0.33 0.83 0.40 0.73 10 0.720.90 0.35 0.88 0.42 0.76 20 0.65 0.81 0.33 0.83 0.40 0.73 40 0.6 0.750.36 0.9 0.38 0.69

TABLE 3 Rabbit No. 1310 Antigen (24) Antigen (23) Antigen (21)Lamotrigine (μg/ml) A₄₀₅ B/Bo A₄₀₅ B/Bo A₄₀₅ B/Bo 0 1.35 1.00 0.60 1.000.7 1.00 2.5 1.30 0.96 0.20 0.33 0.3 0.43 5 1.20 0.89 0.21 0.35 0.3 0.4310 1.15 0.85 0.25 0.42 0.3 0.43 20 1.00 0.74 0.23 0.38 0.28 0.4 40 0.990.73 0.23 0.38 0.27 0.39

B is the absorbance value at 405 nm for the test sample, and Bo is theabsorbance value at 405 nm in the absence of a competing analyte (A405 @zero calibrator), wherein Bo is B at zero calibrator, or no analyte.B/Bo indicates inhibition upon addition of competing analyte (e.g.,ELISA format). The absorbance (B) is dependant on immunoreactions andreaction conditions (buffer, reaction times, parameters, etc). Higherabsorbance can be attributed by strong immunoreactions when an antigenbinds strongly to an antibody strongly, which can be by adding morereagents, by changing of assay parameter, or by changing reaction time,or by changing reaction temperature.

A known amount of analyte, which is a calibrator having a knownconcentration of 0-40 μg/ml competes with lamotrigine analog on theELISA plate for anti-lamotrigine antibody. The absorbance (B) at a givenanalyte concentrate can be lower than the absorbance free of analytewhen the B/Bo is less than or equal to 1. If free drug or analyte bindsstrongly with the antibody, it can freely compete with antigen for theavailable antibody. Absorbance B can then drop quickly as the availableantibody binds only with free drug, which also results in B/Bo decreasequickly. If the binding between free drug and antibody is week, the freedrug does not displace the antigen from the antibody, and the absorbanceB may slightly drop. The B/Bo decreases from 1.00 over the span ofcalibrator range, wherein the changes in B/Bo over the span ofcalibrator range are dependant of immunoreactions between the antigen,free drug, and antibody. The difference in an immunoreaction (e.g.,competition or B/Bo) at various concentrations is essential incompetitive immunoassays. The larger difference in B/Bo over the span ofassay range at each calibrator concentration can result in a moreaccurate measurement. Over the span of assay calibration range, B/Bo isdependant on antigen and antibody interactions. High absorbance (e.g.,OD between 0.5 to 1.5) and large differences in B/Bo at each level ofknown analyte concentration (calibrator) are important in reliable andreproducible immunoassay. A large dynamic range of B/Bo (e.g., largedifference of B/Bo over incremental changes in analyte concentration)over the assay range and a strong absorbance (B) is required to obtainthe accuracy and precision for a commercial immunoassay.

In tables 2 and 3 (e.g., Rabbit 1309), the antigen (24) provides highestabsorbance (B or Bo), and B/Bo decreases over the span of calibratorrange. The Antigen (24) has a similar structure as the immunogen (20),where only carrier protein is different. Due to the similarity instructure, the epitope on the antigen is the same as the epitopestimulated antibody response, and the antibody can recognize the linkeras well as drug. Thus, highest absorbance is expected because it takesmore free drugs to compete with antigen for antibody, and leads to smalldecrease in B/Bo over the span of assay range.

Antigen (23) has a different structure than immunogen (e.g., differentlinker, different site of derivatization, and different carrierprotein), which results in the absorbance (B) being the lowest. Antigen(21) has a different structure than immunogen (e.g., different linker,same site of derivatization, and different carrier protein). Theabsorbance (B) falls in the middle, which is different from theimmunogen, and the differences in B/Bo over span of assay range are thelargest. However, the antigens (24), (23), (21) are not optimal againstthe antibody due to poor titer and poor B/Bo profile.

Example 29

A polyclonal antibody that binds with lamotrigine was prepared using aprotocol similar as described in Example 25. More particularly, thelamotrigine immunogen (18) having the KLH immunogenic moiety was used togenerate the anti-lamotrigine polyclonal antibody.

Example 30

ELISA plates for use in an ELISA assay were prepared substantially inaccordance with Example 26 in order to study the polyclonal antibodyprepared as described in Example 27. More particularly, the antigens(24), (23), (21), (25), and (26) were coated onto ELISA plates.

Example 31

The antibody titer for a polyclonal antibody prepared in accordance withExample 29 was determined using ELISA plates as prepared as in Example30. The protocol for determining the antibody titer was followed asdescribed in Example 27. Accordingly, the plate was read at 405 nm, andthe titer results are provided in Table 4.

TABLE 4 ELISA titer Rabbit Antigen Antigen Antigen Antigen Antigen No.Immunogen 24 23 21 25 26 2689 18 1:240 1:160000 1:800 1:400 1:400 269018 1:100 1:45000  1:200 1:400 1:300These results indicate that the titer was not sufficient for themicroparticle agglutination immunoassay because the microparticlesagglutination immunoassay should be conducted with a titer of at least1:100,000. However, the titer with respect to antigen (23) wassignificantly higher in comparison with the antibodies generated withimmunogen (20). In part, the high titer with respect to antigen (23) canbe attributed to the similarity of the chemical constructs of antigen(23) in comparison with immunogen (18), which are only different by theKLH conjugate being substituted with BSA. As such, it is possible theanti-lamotrigine antibody generated with the immunogen (18) may besuitable for use in commercial immunodiagnostic assay protocols withantigen (23) as the competitor.

Example 32

The avidity of the anti-lamotrigine antibodies prepared with immunogen(18) for lamotrigine analogs were determined by a binding inhibitionstudy performed with a protocol substantially similar as described inExample 28. More particularly, the antigens (23) and (21) were used. Theplate was read at 405 nm, and the results are provided in Tables 5 and6.

TABLE 5 Rabbit No. 2689 Antigen 23 Antigen 21 Lamotrigine (μg/ml) A₄₀₅B/Bo A₄₀₅ B/Bo 0 0.50 1.00 0.80 1.00 2.5 0.30 0.60 0.45 0.56 5 0.01 0.020.35 0.44 10 0.01 0.02 0.30 0.38 20 0.01 0.02 0.20 0.25 40 0.01 0.020.15 0.19

TABLE 6 Rabbit No. 2690 Antigen 23 Antigen 21 Lamotrigine (μg/ml) A₄₀₅B/Bo A₄₀₅ B/Bo 0 0.75 1.00 0.5 1.00 2.5 0.05 0.07 0.25 0.50 5 0.05 0.060.28 0.56 10 0.02 0.03 0.2 0.40 20 0.02 0.03 0.18 0.36 40 0.01 0.01 0.160.32

The immunogen (20) has a short linker, and is not as immunogenic asimmunogen (19). The antibody produced from immunogen (20) shows titer aslow as 1: 210 and as high as 1:11000, and has a change in B/Bo over thespan of assay range being too small or too large. The immunogen (18) hasa short linker, and is not as immunogenic as immunogen (19). Theantibody produced from immunogen (18) shows good titer (e.g., Rabbit2690 titer 1:45,000; Rabbit 2689 titer 1:160,000) against antigen (23),but low titer against all other antigens.

Antigen (23) has the similar structure as the immunogen (18) (e.g., onlycarrier protein is different). Due to the similarity in structure, theepitope on the antigen is the substantially the same as the epitopestimulated antibody response. As such, the antibody can recognize thelinker as well as the drug, and the highest absorbance is observed. Theimprovement of titer in the antibody from rabbits 2689 and 2690, whichwas up to 1:160,000 and 1:45,000, respectively, may be caused by theincreased recognition of the antigen by the antibody.

The incremental changes in B/Bo over the span of assay range showspromising B/Bo profile. Antigen (21) is a good competitor againstlamotrigine for the antibody when only B/Bo is considered. The antibodyshows a low titer (poor recognition) against antigen (21), which may bebecause this antigen has a different structure than immunogen (18)(e.g., different linker, same site of derivatization, and differentcarrier protein).

Example 33

A polyclonal antibody that binds with lamotrigine was prepared using aprotocol similar as described in Example 25. More particularly, thelamotrigine immunogen (19) having the KLH immunogenic moiety was used togenerate the anti-lamotrigine polyclonal antibody.

Example 34

ELISA plates for use in an ELISA assay were prepared substantially inaccordance with Example 26 in order to study the polyclonal antibodyprepared as described in Example 33. More particularly, the antigens(24), (23), (21), (25), and (26) were coated onto ELISA plates.

Example 35

The antibody titer for a polyclonal antibody prepared in accordance withExample 33 was determined using ELISA plates as prepared as in Example34. The protocol for determining the antibody titer was followed asdescribed in Example 27. Accordingly, the plate was read at 405 nm, andthe titer results are provided in Table 7.

TABLE 7 Rabbit ELISA titer No. Immunogen Antigen 24 Antigen 23 Antigen21 Antigen 25 Antigen 26 2693 19 1:300000 1:310000 1:700000 1:44000001:1900000 2694 19 1:200000 1:1700000 1:850000 1:4400000 1:1800000

The anti-lamotrigine polyclonal antibodies prepared with immunogen 19,which includes a long linker between the lamotrigine scaffold and theimmunogenic moiety, exhibit high titers that are suitable formicroparticle agglutination immunoassays. As such, long linkers can bebeneficial for imparting efficacious immunogenicity to an immunogenbased on the lamotrigine drug. As such, the linker of immunogen 19, orthose having similar length or other property, can be conjugated at the4-position and the 3-position of lamotrigine analogs.

Example 36

The avidity of the anti-lamotrigine antibodies prepared with immunogen(19) for lamotrigine analogs were determined by a binding inhibitionstudy performed with a protocol substantially similar as described inExample 26. More particularly, the antigens (24), (23), (21), (25), and(26) were used. The plate was read at 405 nm, and the results areprovided in Tables 8 and 9.

TABLE 8 Antigen Antigen Antigen Antigen Antigen Rabbit No. 2693 24 23 2125 26 Lam. (μg/ml) A₄₀₅ B/Bo A₄₀₅ B/Bo A₄₀₅ B/Bo A₄₀₅ B/Bo A₄₀₅ B/Bo 00.28 1.00 0.68 1.00 1.55 1.00 0.32 1.00 1.1 1.00 2.5 0.08 0.29 0.08 0.121.52 0.98 0.16 0.50 0.4 0.36 5 0.05 0.18 0.08 0.12 1.45 0.94 0.15 0.470.28 0.25 10 0.04 0.14 0.08 0.12 1.40 0.90 0.08 0.04 0.24 0.22 20 0.030.11 0.08 0.12 1.28 0.83 0.07 0.22 0.20 0.18 40 0.02 0.07 0.08 0.12 1.200.77 0.07 0.22 0.16 0.15

TABLE 9 Antigen Antigen Antigen Antigen Antigen Rabbit No. 2693 24 23 2125 26 Lam. (μg/ml) A₄₀₅ B/Bo A₄₀₅ B/Bo A₄₀₅ B/Bo A₄₀₅ B/Bo A₄₀₅ B/Bo 00.83 1.00 0.44 1.00 1 1.00 0.72 1.00 1.80 1.00 2.5 0.28 0.34 0.08 0.180.90 0.90 0.36 0.50 1.16 0.64 5 0.22 0.27 0.08 0.18 0.85 0.85 0.32 0.441.04 0.58 10 0.15 0.18 0.08 0.18 0.80 0.80 0.28 0.39 0.88 0.49 20 0.120.14 0.08 0.18 0.78 0.78 0.20 0.28 0.80 0.44 40 0.10 0.12 0.08 0.18 0.700.70 0.18 0.25 0.68 0.38

Immunogen (19) has a long linker and the epitope is more accessbile forantibody interaction, and was the most immunogenic with the antibodyhaving the highest titer of up to 1:4,400,000. All antigens (e.g., 21,23, 24, 25, 26) show fair to good competition with lamotrigine free drugfor the polyclonal antibody produced from immunogen (19).

Antigen (24) has a short linker and the site of derivatization is thesame as immunogen (19). The weaker recognition of antibody by antigen(24) leads to relatively less titer of up to 1:300,000. The binding ofantigen (24) and antibody is comparatively weaker due to less availablesurface area for antibody interaction. The B/Bo shows sharp decreaseeven at very small concentrations of lamotrigine, where incrementalchanges in B/Bo at higher lamotrigine concentrations shows thefeasibility of antigen (24).

Antigen (23) has a short linker and the site of derivatization isdifferent from immunogen (19). The comparatively weaker recognition ofantibody by antigen (23) leads to relatively lower titer of up to1:700,000. The binding of antigen (23) and antibody is very weaker dueto less available surface area and less recognition for antibodyinteraction. The B/Bo shows sharp decrease even at very smallconcentration of lamotrigine, which indicates a preference forlamotrigine instead of the lamotrigine analog.

Antigen (21) has a similar structure as the immunogen (19), excepthaving a different carrier protein. The antibody shows moderate titeragainst antigen (21).

Antigen (25) has a shorter but similar linker than immunogen (19). Infact, the analog (6) used in the preparation of antigen (25) is theprecursor of analog (8), which is used in the preparation of immunogen(19). Strong recognition resulted from the similarity in structure leadsto very high titer of up to 1:4,400,000. Antigen (25) freely competesagainst free drug for antibody due to the difference in structure, andshown incremental changes in B/Bo. Antigen (25) and the polyclonalantibody could be used in commercial immunoassay because of the hightiter and good B/Bo profile.

Antigen (26) has a very long linker, which is second longest next toantigen (21). The increased immureaction between antigen (25) andantibody leads to high titer of up to 1,900,000. Antigen (26) freelycompetes against free drug for antibody due to the difference instructure, and shown incremental changes in B/Bo. Antigen (26) and thepolyclonal antibody are currently used in commercial immunoassay becauseof high titer and good B/Bo profile. Thus, the anti-lamotriginepolyclonal antibodies prepared with immunogen 19 were shown to havefavorable avidity, and showed competitive binding profiles suitable forimmunodiagnostic assays.

Example 37

The antibody titer for a polyclonal antibody prepared in accordance withExample 33 was determined using ELISA plates as prepared similarly as inExample 34; however, anti-rabbit IgG and anti-rabbit IgM were coatedonto the ELISA plate. The protocol for determining the antibody titerwas followed as described in Example 27, except antigen 21 was used.Accordingly, the plate was read at 405 nm, and the titer results areprovided in Table 10.

TABLE 10 Subclass IgG/IgM ELISA test Titer, against antigen 20 RabbitNo. Anti-rabbit IgG Anti-rabbit IgM 2693 1:1700000 1:70  2694 1:35000001:100

The titer shows that both anti-rabbit IgG and anti-rabbit IgM wereactivated against the antigen. The polyclonal antisera are mostly IgGantibodiess due to the fact that the antibodies show much higher titeragainst anti-rabbit IgG.

Example 38

Anti-lamotrigine antibodies that bind with lamotrigine and analogs wereprepared for use in immunodiagnostic assays. The monoclonal antibodieswere prepared in nine female Balb/C mice that were 16 weeks of age orolder, which were immunized by multiple injections of immunogen (19).The immunogenic injection solution for each mouse included 250 μL of animmunogenic solution comprising immunogen (19), which was mixed with 250μL of complete Freund's adjuvant. The immunogenic injection solution wasloaded into an appropriately sized syringe fitted with a 37 gaugehypodermic needle and injected into each mouse. The booster injectionswere repeated after 14 days, but using incomplete Freund's adjuvant. Thebooster injections were repeated again on day 60 and day 80.Additionally, on day 45 the mice were tested for anti-lamotrigineantibody by acquiring blood via a tail bleed, wherein the antibodieswere tested by ELISA to determine titer and avidity.

Example 39

ELISA plates for use in an ELISA assay were prepared substantially inaccordance with Example 26 in order to study the antibody prepared asdescribed in Example 38. More particularly, the antigen (21) was coatedonto ELISA plates. Briefly, antigen (21) was diluted in a coating bufferand was added to the wells of the ELISA plate. After being incubated for60 min at 37° C., the buffer solvent was decanted and a blocking bufferwas added to the plates. The plate was again incubated for 60 min at 37°C. and the blocking solvent was decanted from the plate. The plate wasthen stored with the blocking agent in the wells at 2-8° C. for up to 1week.

Example 40

The anti-lamotrigine antibody titer was determined using the bloodobtained from the tail bleed described in Example 36. The titerdetermination protocol was initiated by the bleeds being seriallydiluted from 1:100 to 1:10,000,000 by using a 10-fold dilution. Thedilutions are prepared in microcentrifuge tubes containing PBS at pH7.4. About 100 μL of sample obtained from the blood was added to eachwell on the ELISA plate. The plate was then incubated for 60 min at 37°C. and washed three times with 250 μL of PBS at pH 7.4 with 0.05% tween.About 125 μL of a diluted second antibody conjugate in PBS at pH 7.4 wasadded to each well of the plate. About 125 μL of ABTS substrate wasadded to each well of the plates and the plate was incubated for 20 minbefore being read at 405 nm, where the results are shown in Table 11.

TABLE 11 Mouse # (ELISA titer, against antigen 21) Dilution 1 2 3 4 5 78 9 10 1:100 3.44 3.50 3.516 3.539 3.541 3.5845 3.3305 3.401 3.44951:1000 3.31 3.28 3.448 3.4095 3.455 3.5885 3.4135 3.211 3.183 1:100003.18 3.20 3.2285 3.2135 3.0945 3.2785 3.0835 2.907 2.9205 1:100000 2.211.24 2.873 2.585 2.5615 2.804 2.7455 2.1635 2.1495 1:1000000 0.62 0.221.178 0.7735 0.848 0.9655 1.0545 0.508 0.518 1:10000000 0.25 0.10 0.24750.1875 0.167 0.2315 0.21 0.1465 0.1255

The titer is calculated by an end point titer having about 10% of themaximum OD. In Table 11, the average maximum OD is 3.5., and theantibody titer is 10% of maximum O.D. of 0.35. Mouse 3 has the highesttiter because at 1:1,000,000 dilution and at 1:10000000 dilution, it hasthe highest absorbance (OD)

Example 41

The avidity of the anti-lamotrigine antibodies prepared with immunogen(19) in accordance with the protocol described in Example 38 forgenerating monoclonal antibodies was determined by a bind inhibitionstudy performed with a protocol substantially similar as described inExample 28. More particularly, the antigen (21) was used. The plate wasread at 405 nm, and the results are provided in Tables 12 and 13.

TABLE 12 Lamotrigine Absorbance (ELISA Avidity) concentration Mouse #(μg/ml) 1 2 3 4 5 7 8 9 10 0 0.6995 0.9205 0.7555 0.8865 0.816 0.46450.6215 0.5515 0.5105 280 0.6965 0.8135 0.693 0.781 0.7055 0.356 0.43050.347 0.4475

TABLE 13 Mouse # 1 2 3 4 5 7 8 9 10 % 0.43 11.62 8.27 11.9 13.54 23.3630.73 37.08 12.34 inhi- bition B/Bo × 99.57 88.38 91.73 88.1 86.46 76.6469.27 62.92 87.66 100

Fusion candidate was chosen based on the titer (e.g., the amount of theantibody in the blood) and avidity (e.g., specificity againstlamotrigine) of the polyclonal antisera. B/Bo indicates inhibition uponaddition of a competing analyate, where B/Bo=100−% inhibition. Table 13shows that polyclonal antibody from mouse No. 1, No. 2, No. 3, No. 4,and No. 10 show preference for the lamotrigine analog (ELISA).Polyclonal antibody from mouse No. 8 and No. 9 show a large differencein B/Bo (or % inhibition) over the span of assay (0-200 μg/ml).

Example 40

A fusion candidate was prepared in order to generate monoclonalantibodies that can bind with lamotrigine and analogs. The fusioncandidate was chosen based on avidity and/or the titer or amount of theantibody in the blood. If sensitivity (e.g., to detect low concentrationof analyate) or specificity (e.g., to differentiate between analyate andcross reactant) are the desired quality of monoclonal antibody, mouseNo. 9 and mouse No. 8 are the prime candidates. Highest titer valuesindicate the highest amount of antibody in the blood, and the successrate is the highest when the antibody concentrate in spleen is highest.Thus, Mouse No. 1 is the candidate for the likely fusion success

The procedure for producing the fusion candidate was conducted by theimmunized mouse being given a final booster injection three to five daysbefore the fusion. This booster injection was administered 4 weeks afterthe previous injection, where this interval can allow most of thecirculating antibodies to be cleared from the blood stream by the mouse.The final booster injection is used for two purposes: (1) to induce agood, strong response; and (2) to synchronize the maturation of theresponse. This can allow an increase in the relative concentration ofthe appropriate B-lymphocyte fusion partners. The final boost was directat the spleen since it is the best choice for lymphocyte isolation. Thespleen of the mouse was removed using aseptic technique and placed in 10mL of complete culture medium in a sterile petri dish, and was thenground between two sterile frosted microscope slides. The resultingsingle-cell suspension was drawn off and counted using a hemocytometer.Myeloma cells were mixed into the spleen cells in a ratio of 1:5 andcentrifuged for 15 min at about 800×G. The supernatant liquid was drawnoff and discarded, and 15 mL of serum-free IMDM culture media was added.The cells were re-suspended and again centrifuged. The resulting cellpellet was fused using polyethylene glycol/DMSO.

After fusion, the cells were diluted in Iscove's Dulbecco's mediumsupplemented with 10% fetal bovine serum (Hyclone Labs), 10%> condimedHI, 50 mM 2-mercaptoethanol, 20 mM ethanolamine,hypoxanthine-methotrexate-thymidine, 4 mM glutamine, and pen/strepantibiotics. This mixture of fused cells was plated at 200 μL/well intosterile 96-well microculture plates. The covered plates were placed inan incubator for 6 days at 37 degree C. in 5% CO₂.

Example 43

The avidity of the anti-lamotrigine antibodies prepared with immunogen(19) in accordance with the protocol described in Example 42 forgenerating monoclonal antibodies was determined by a binding inhibitionstudy performed with a protocol substantially similar as described inExample 26. More particularly, antigen (21) was used. The plate was readat 405 nm, and the results are provided in Tables 14 and 15.

TABLE 14 Absorbance (ELISA) Lamotrigine Clones (after Fusion)concentration (μg/mL) 1D11 3E8 4G6 5G11 8B10  4B11 7E10 0 1 0.14851.7225 1.333 1.2675 1.07 1.2665 280 0.9475 0.126 0.201 0.853 0.755 0.6360.823

TABLE 15 Clones (after fusion screening) 1D11 3E8 4G6 5G11 8B10 4B117E10 % inhibition 5.25 15.15 88.33 36.01 40.43 40.56 35.02 B/Bo × 10094.75 84.85 11.67 63.99 59.57 59.44 64.98

The Inhibition profile from Table 15 shows the fused clone 4G6 prefersfree drug (e.g., large % inhibition or smallest B/Bo) and the fusedclone 3E8 prefers the lamotrigine analog (21).

Example 44

After fusion, as described in Example 43, 1× clones were prepared.Accordingly, after a positive tissue culture supernatant has beenidentified, the next step is to clone the antibody-producing cell. Theoriginal positive well often contains more than one clone of hybridomacells, and many hybrid cells have an unstable assortment of chromosomes.Single-cell cloning ensures that cells that produce the antibody ofinterest are truly monoclonal and are stable. The hybridoma cells werecloned by limiting dilutions. A growth medium was added to a wellcontaining fused cells. The clones grew rapidly and the 1× clones wereready for screening (ELISA and QMS) after two weeks. The avidity of theanti-lamotrigine antibodies was determined by a binding inhibition studyperformed with a protocol substantially similar as described in Example28. More particularly, antigen (21) was used. The plate was read at 405nm, and the results are provided in Tables 16 through 21.

TABLE 16 Absorbance (ELISA) Lamotrigine 1X Clones concentration 1D11-1D11- 1D11- 3E8- 3E8- 3E8- 4G6- (μg/mL) 1 10 30 7 14 25 5 0 2.123 2.332.05 2.13 2.21 2.27 1.87 200 0.475 1.12 0.38 0.67 0.455 0.814 0.39

TABLE 17 1X Clones 1D11- 1D11- 1D11- 3E8- 3E8- 3E8- 4G6- 1 10 30 7 14 255 % inhibition 77.6 51.9 81.5 68.4 79.4 64.2 78.7

TABLE 18 Lamotri- gine Absorbance (ELISA) concentra- 1X Clones tion 4G6-4G6- 5G11- 5G11- 5G11- 8B10- 8B10- (μg/mL) 21 28 7 18 34 9 14 0 2.052.08 2.24 2.15 2.38 1.98 1.68 200 0.34 0.31 1.17 0.7015 1.39 0.22 0.42

TABLE 19 1X Clones % 4G6- 4G6- 5G11- 5G11- 5G11- 8B10- 8B10- inhibition21 28 7 18 34 9 14 0 83.1 85 47 67 42 89 75

TABLE 20 Absorbance (ELISA) Lamotrigine 1X Clones concentration 4B11-7E10- 7E10- 7E10- (μg/mL) 8B10-23 4B11-3 4B11-11 17 8 26 37 0 2.03 1.891.92 2.61 2.25 2.36 2.45 200 1.02 0.24 0.24 0.56 0.96 0.85 0.60

TABLE 21 % in- 1X Clones hibi- 8B10- 4B11- 4B11- 4B11- 7E10- 7E10- 7E10-tion 23 3 11 17 8 26 37 0 49.5 87.1 87.7 78.4 57.2 64 75.4

The larger percent inhibition indicates that the clone prefers the freelamotrigine over the lamotrigine analog (21).

A QMS® assays was performed to test the 1× clones prepared as in Example44. The screening on QMS® is not optimized (assay parameter and titer ofthe 1× clones). The data is shown on Table 22.

TABLE 22 Lamotrigine Delta Absorbance (Immunoturbidimetric Format)concentration 1X Clones (μg/mL) 4G6-21 4G6-28 5G11-34 0 1.1254 0.78300.2172 40 0.0024 0.0013 0.0000 % inhibition 99.8 99.8 99.9

It can be observed from the Table 22 that the 1× clones recognize thelamotrigine analog (21), and the ability of lamtorigine free drug toinhibit the immunoreaction indicates the binding is specific tolamotrigine as opposed to non-specific binding of protein carrier. Thus,the clones may be used in an immunoassay.

Example 45

The QMS® lamotrigine assay is an automated homogeneous particle-enhancedturbidimetric immunoassay used for the analysis of lamotrigine in serumor plasma. A QMS® assay was performed to test the polyclonal antibodiesprepared as in Example 34. The QMS assay for lamotrigine was conductedusing a liquid, ready-to-use, two-reagent kit, which contains: R1, whichis comprised of sheep polyclonal antibodies that bind with lamotrigineprepared from immunogen (19) at less than <1% in bis-tris buffer withabout sodium 0.05% azide; and R2, which is comprised oflamotrigine-coated microparticles with antigen (26) at less than 0.5%with sodium azide at 0.05%. The QMS lamotrigine assay can be calibratedusing a full calibration (6-point) procedure to generate a calibrationcurve similar to FIG. 4, wherein Seradyn QMS lamotrigine calibrators(0.0, 2.5, 5, 10, 20, 40 μg/ml) are used. The results are provided inTable 23.

TABLE 23 Polyclonal antibody R1 Rate (Delta Absorbance) Lamotrigine(μg/mL) sample Lamotrigine antigen (22) coated Latex 0 179 2.5 107 5 6010 34 20 18 40 11

When a sample containing lamotrigine is added, the agglutinationreaction is partially inhibited and observed by slowing down the rate ofabsorbance change. A s such, a concentration-dependent classicagglutination inhibition curve can be obtained with maximum rate ofagglutination at the lowest lamotrigine concentration (e.g., at zeroμg/ml) and the lowest agglutination rate at the highest lamotrigineconcentration (e.g., 40 μg/ml). The incremental changes in rate over theassay range shown in Table 23 indicate the antibody, antigen(competitor), and the free drug (lamotrigine) interaction is suitablefor a commercial immunoassay for use in an automated system.

Example 46

An experiment was performed to compare an automated homogeneousparticle-enhanced turbidimetric immunoassay to an HPLC method fordetecting lamotrigine. The method comparison assay is an experimentdesigned to evaluate the bias between two methods that measure the sameanalyate. A QMS® assay was performed as described in Example 45 to testthe polyclonal antibodies prepared as in Example 34. As such,lamotrigine patient samples are assayed and compared to a referencemethod HPLC. The purpose of the evaluation is to determine if the twomethods yield equivalent results within statistical power of theexperiment. The comparison experiment was conducted with twenty fivepatient samples consisting of serum or sodium heparinized plasma.Concentrations in the turbidimetric immunoassay ranged from 1.59 to 35μg/mL and concentrations on the HPLC ranged from 1.3 to 32.9 μg/mL.Results from the automated QMS® lamotrigine assay were compared with theresults from HPLC and shown in Tables 23 and 24.

TABLE 23 Seradyn HPLC Seradyn QMS (H717) ID # Result Rep 1 Rep 2 Mean0001 4.2 5.14 5.16 5.2 0002 1.3 1.6 1.57 1.6 0003 7.7 8.37 8.29 8.3 00049.6 8.92 8.87 8.9 0005 1.8 1.95 1.97 2.0 0006 10.3 12.63 13.33 13.0 00078.2 9.08 8.91 9.0 0008 9.1 10.22 10.11 10.2 0009 2.4 2.66 2.75 2.7 001015.6 16.93 16.38 16.7 0011 5.5 6.13 5.91 6.0 0012 19.8 21.56 21.45 21.50013 12.7 14.55 14.48 14.5 0014 12.3 13.73 13.99 13.9 0015 21.2 24.1324.36 24.2 0016 16.7 18.68 18.9 18.8 0017 3.1 3.29 3.44 3.4 0018 3.64.33 4.27 4.3 0019 2.8 3.41 3.36 3.4 0020 6.7 7.98 7.72 7.9 0021 13.715.31 15.55 15.4 0022 11.1 12.93 13.24 13.1 0023 18.1 18.32 18.75 18.50024 18.8 25.35 22.45 23.9 0025 32.8 38.37 32.88 35.6

TABLE 24 Method HPLC n 25 Y-intercept 0.0151 Slope 1.107 Correlation0.994 Coefficient

As shown in FIG. 18, the difference between the automated homogeneousparticle-enhanced turbidimetric immunoassay and the HPLC method wasdetermined to be less than about 10%. As can be seen in FIG. 19, theconcentrations in the turbidimetric immunoassay ranged from 1.59 to 35μg/mL and concentrations on the HPLC ranged from 1.3 to 32.9 μg/mL. Theslope of the QMS® lamotrigine assay on the turbidimetric immunoassay was1.107 with an intercept of 0.0151, which was compared to the HPLC methodprepared by ARUP (Salt Lake City Utah) HPLC/UV. The correlationcoefficient (R) was 0.994. Bias plot shows 10% bias between HPLC and QMSvalues. Thus, the data shows that QMS® assay is a suitable replacementfor HPLC reference method.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed is:
 1. An antibody composition for use in animmunodiagnostic assay for detecting the presence of lamotrigine in asample from a patient treated with lamotrigine, the compositioncomprising: an anti-lamotrigine antibody produced by an immunogeniccomposition including a lamotrigine derivative, the lamotriginederivative having a chemical structure of Formula 1;

wherein: L is NH; W is CO(CH₂)₂CONH(CH₂)₂; X is NHCO(CH₂)₆; Y is Y₁—Z,wherein Y₁ is CO and Z is BSA or KLH; and wherein the anti-lamotrigineantibody has at least one binding domain capable of binding lamotrigine,and wherein the antibody has a titer at least 1:100,000.
 2. The antibodycomposition as in claim 1, wherein the antibody is a monoclonalantibody.
 3. The antibody composition as in claim 1, wherein theantibody is a polyclonal antibody.
 4. A polyclonal or monoclonalantibody composition for use in an immunodiagnostic assay for detectingthe presence of lamotrigine in a sample, the polyclonal or monoclonalantibody composition comprising: an anti-lamotrigine antibody producedby an immunogenic composition including a lamotrigine derivative, thelamotrigine derivative having a chemical structure of Formula 1;

wherein: L is NH; W is CO(CH₂)₂CONH(CH₂)₂; X is NHCO(CH₂)₆; Y is Y₁—Z,wherein Y₁ is CO and Z is BSA or KLH; and the anti-lamotrigine antibodyhaving at least one binding domain capable of binding lamotrigine, andwherein the antibody has a titer at least 1:100,000.
 5. The polyclonalor monoclonal antibody composition as in claim 4, wherein the antibodyis a monoclonal antibody.
 6. The polyclonal or monoclonal antibodycomposition as in claim 4, wherein the antibody is a polyclonalantibody.
 7. A method for producing an anti-lamotrigine antibody,comprising: administering at least a first dose of an immunogeniccomposition including a lamotrigine derivative to an antibody producingsubject, the lamotrigine derivative having a chemical structure ofFormula;

wherein: L is NH; W is CO(CH₂)₂CONH(CH₂)₂; X is NHCO(CH₂)₆; Y is Y₁-Z,wherein Y₁ CO and Z is BSA or KLH; and collecting the antibodies fromthe antibody producing subject; and purifying and/or screening theantibodies collected from the antibody producing subject.
 8. The methodof claim 7, further comprising administering to the antibody producingsubject at least a second dose of the immunogenic composition prior tocollecting the antibody from the antibody producing subject.
 9. Themethod of claim 7, wherein the collecting includes at least one ofobtaining blood, serum, plasma, or other biological sample from theantibody producing subject.
 10. The method of claim 7, wherein thescreening includes and ELISA assay.